Biomarkers for the early detection of autoimmune diseases

ABSTRACT

Provided are methods for aiding in diagnosing autoimmune diseases in a mammal, comprising contacting a sample of tear from the mammal with an antibody that specifically binds to a first polypeptide selected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il16ra, Il10, Il10ra, Il15, Tnfa, Apo-F, or Lcn-2 or a second polypeptide selected from the group lactoperoxidase, lactoferrin or lysozyme under conditions favoring the formation of an antibody-polypeptide complex, and determining the amount of complex formed, wherein an increased formation of antibody-first-polypeptide complex or a decreased formation of antibody-second-polypeptide complex as compared to a suitable control, indicates a likely positive diagnosis of an autoimmune disease for the mammal, thereby aiding in the diagnosis. Methods of treating the autoimmune diseases are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. Nos. 61/328,168, filed Apr. 26, 2010;61/266,985, filed Dec. 4, 2009 and 61/223,692, filed Jul. 7, 2009.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under the Contract No.RO1 EY011386, EY017293 and EY016985 awarded by the National Institutesof Health. The government has certain rights to the invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for the diagnosis ofautoimmune diseases and for their treatments.

BACKGROUND

Autoimmune diseases arise from an overactive immune response of the bodyagainst substances and tissues normally present in the body in which thebody actually attacks its own cells. The immune system mistakes somepart of the body as a pathogen and attacks it. This may be restricted tocertain organs (e.g. in thyroiditis) or involve a particular tissue indifferent places (e.g. Goodpasture's disease which may affect thebasement membrane in both the lung and the kidney). The treatment ofautoimmune diseases is typically with immunosuppression—medication whichdecreases the immune response. The mechanisms of autoimmune diseases arenot well understood and the treatment options are limited.

Examples of autoimmune diseases include Coeliac disease, diabetesmellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Sjögren'ssyndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves'disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis(RA).

Sjögren's syndrome (SjS) is a chronic autoimmune inflammatory diseasecharacterized by lymphocytic infiltration and destruction of lacrimalglands (LG) and salivary gland function (SG). SjS can occurindependently (primary SjS) or in conjunction with another autoimmunedisease (secondary SjS); both forms may progress to systemic disease ofother organs. In both primary and secondary SjS, the presenting symptomsof ocular surface dryness, corneal irritation and increasedsusceptibility to infection overlap with symptoms of simplekeratoconjunctivitis sicca (KCS). Despite the potentially unique diseaseprofile that is likely to be manifested in the tears of primary andsecondary SjS patients, no tear biomarkers have been established asdiagnostic for either form.

Definition of tear biomarkers which can predict disease severity wouldbe valuable diagnostically in combination with existing clinicalstrategies, potentially contributing to different choices of therapy andimproved disease outcome for SjS as well as for other autoimmunediseases. Since sensitivity of detection and stability of biomarkers anddetection reagents are significant challenges given the limited samplesizes and extensive protease content of even normal tears, developmentof alternative detection strategies for tear biomarkers is clearlywarranted. This invention satisfied this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

One aspect of the invention provides a method for determining whether amammal is likely to develop autoimmune disease, comprising, oralternatively consisting essentially of or yet further consisting ofmeasuring an expression level or activity level of at least one firstpolypeptide selected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng,Il6ra, Il10, Il10ra, Il15, Tnfa, or Lcn-2, and/or at least one secondpolypeptide selected from the group lactoperoxidase, lactoferrin orlysozyme in a sample of tears isolated from the mammal, wherein anincreased expression level or increased activity level of the firstpolypeptide or a decreased expression level or decreased activity levelof the second polypeptide, as compared to a suitable control, indicatesthat the mammal is likely to develop autoimmune disease. In a furtheraspect, measuring the expression level or activity level of Apo-F isexcluded from the method.

Also provided is a method for aiding in the diagnoses of autoimmunedisease in a mammal, comprising, or alternatively consisting essentiallyof or yet further consisting of measuring an expression level oractivity level of at least one first polypeptide selected from the groupCtss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10, Il10ra, Il15, Tnfa,Apo-F, or Lcn-2, and/or at least one second polypeptide selected fromthe group lactoperoxidase, lactoferrin or lysozyme in a sample of tearisolated from the mammal, wherein an increased expression level orincreased activity level of the first polypeptide or a decreasedexpression level or decreased activity level of the second polypeptide,as compared to a suitable control, indicates a likely positive diagnosisof autoimmune disease for the mammal, thereby aiding in the diagnosis.In a further aspect, measuring the expression level or activity level ofApo-F is excluded from the method.

Further provided is a method for diagnosing relative severity ofautoimmune disease in a mammal, comprising, or alternatively consistingessentially of or yet further consisting of measuring an expressionlevel or activity level of at least one first polypeptide selected fromthe group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10, Il10ra, Il15,Tnfa, Apo-F, or Lcn-2, or at least one second polypeptide selected fromthe group lactoperoxidase, lactoferrin or lysozyme in a sample of tearfrom the mammal, wherein a relatively higher expression level oractivity level of the first polypeptide or a relatively lower expressionlevel or activity level of the second polypeptide, as compared to asuitable control, indicates that the individual has relatively moresevere autoimmune disease. In a further aspect, measuring the expressionlevel or activity level of Apo-F is excluded from the method.

In some embodiments of the methods of the invention, the firstpolypeptide is one or more of Ctss, Apo-F or Lcn-2. In a further aspect,measuring the expression level or activity level of Apo-F is excludedfrom the method.

Another aspect of the invention provides a method of treating a mammalsuffering from or at risk of developing autoimmune disease andidentified as being in need of such treatment by any one of the methodsprovided above, comprising, or alternatively consisting essentially ofor yet further consisting of administering an effective amount of asuitable therapy to the mammal, thereby treating the mammal.Non-limiting examples of suitable therapies include administration of aneffective amount of cyclosporin, cevimeline, pilocarpine, a nonsteroidalanti-inflammatory drug, a corticosteroid, an immunosuppressive drug or adisease-modifying antirheumatic drug. In one aspect, the mammal is ahuman patient.

In another aspect, the invention provides a method for treating a mammalsuffering from or at risk of developing autoimmune disease, comprising,or alternatively consisting essentially of or yet further consisting ofadministering to the mammal an effective amount of an agent inhibitingthe expression or activity of one or more of a polypeptide selected fromCtss, Ctsh, Ctsr, Ctsw or Ctsz. In one aspect, the polypeptide is Ctss.In one embodiment, the mammal is a human patient.

Autoimmune diseases that can be diagnosed or treated by the methods ofthe invention include, without limitation, Coeliac disease, diabetesmellitus type 1 (IDDM), lupus erythematosus, systemic lupuserythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome,Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenicpurpura, rheumatoid arthritis (RA), ankylosing spondylitis, Crohnsdisease, dermatomyositis, Goodpasture's syndrome, Guillain-Barrésyndrome (GBS), mixed Connective tissue disease, multiple sclerosis,myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anaemia,psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis,relapsing polychondritis, temporal arteritis, ulcerative colitis,vasculitis and Wegener's granulomatosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1D depict validation of microarray data and expandedinvestigation for genes of interest by real-time RT-PCR in LG from NOD,NOD SCID and BALB/c mice. Differentially expressed genes in LGs of NODand BALB/c mice, suggested by microarray analysis, encoding Ctsh, Ctss,Ctsz and macrophage-produced cytokines were validated by real-timeRT-PCR in LGs from 12-week-old NOD mice, matched BALB/c control, andmore animal groups as shown in the figure panels. Certain relevantcytokines that were not detected by microarray due to its relativeinsensitivity were also re-evaluated in this study. Triplicates of eachreaction were set up in parallel. The results were repeated 2-3 timesand reproduced with RNAs from different batches of animals. Thecomparisons between different samples were conducted using theformulation of ΔΔCt study built into the ABI SDS 2.1 software. Theexpression level of all the genes in 12-week-old male BALB/c mice weredesignated as 1.0, and the expression levels of these genes in the restof mice were compared to that in 12-week-old male BALB/c mice.

FIGS. 2A and 2B show the detection of CATS in different locations in LGfrom different mouse strains. Cryosections of LGs from 12-week-old maleNOD, NOD SCID and BALB/c mice were incubated with goat anti-mouse CATSpolyclonal antibody and rat anti-CD68 monoclonal antibody followed byappropriate fluorophore-conjugated secondary antibodies. The sectionswere imaged by confocal fluorescence microscopy. Nuclei were stainedwith DAPI and actin filaments with Alexa Fluor 647 in all panels todelineate the relative cellular location of the positive signals.Arrowheads point to CATS-positive cells in the surrounding region of theLG; arrows to CATS-positive cells in the interior region of the LG;hollow arrowheads to CATS- and CD68-positive cells in the surroundingregion of the glands; and arrows to CATS and CD68-double positive cellsin the interior region of the gland. Bars=10 μm

FIG. 3 illustrates the redistribution of CATS protein in LG acinar cellsfrom NOD and NOD SCID mice. Cryosections of LGs from 12-week-old maleNOD, NOD SCID and BALB/c mice were incubated with goat anti-mouse CATSpolyclonal antibody and rat anti-Lamp2 monoclonal antibody followed byappropriate fluorophore-conjugated secondary antibodies. The sectionswere imaged by confocal fluorescence microscopy. Lamp2-enriched vesiclesmarked late endosomes/lysosomes. Labeling with DAPI for nuclei and AlexFluor 647 for actin filaments was conducted to delineate the relativelocation of the targets. Arrowheads point to the CATS-positive areas;arrows to the Lamp2-positive areas. Bars=10 μm.

FIGS. 4A and 4B show comparison of CATS abundance and activity of LGbetween NOD and BALB/c mice. A: Western blotting to compare the proteinabundance of CATS. LG lysates were prepared from 12-week-old male NODmice or matched BALB/c mice. 100 μg each of LG lysates was loaded ineach well of a 11% SDS-polyacrylamide gel. 50 μg of Raw264.7 cell lysatewas run in parallel as a positive control. The proteins transferred tonitrocellular membrane were hybridized with goat anti-CATS polyclonalantibody. One of the two membranes prepared in parallel was hybridizedwith rabbit anti-Rab3D antibody as a loading control; this protein ishighly abundant in LG. B: CATS activity assay. Right: 10 μg each ofpaired LG lysates from the two strains were incubated for 1, 2 and 18hours and the fluorescence of the products was measured atexcitation/emission wave lengths of 400/505 nm. The enzymatic activityis expressed as fluorescent units; error bars show SEM. Left: Assayswere conducted as described with the addition of CATS inhibitor to thereactions to verify the specificity of the enzyme in the LG lysates.

FIGS. 5A and 5B show comparison of CATS enzymatic activities fromstimulated LG lysates and tear fluid between NOD and BALB/c mice. Micewere anesthetized and tear fluid collected following stimulation withCCH as described in Materials and Methods. Catalytic activity wasassayed in the absence or presence of the specific CATS inhibitor usedin FIG. 4. A: CATS activity in stimulated glands and average fold changevalues based on pairwise comparisons. B: CATD activities in stimulatedLG and tears between the paired NOD and BALB/c mice. Errors depict SEM.

FIGS. 6A through 6L show detection of CATH positive cells in differentlocations in LG from different mouse strains. Cryosections of LG from 12week male NOD, NOD SCID and BALB/c mice were incubated with goatanti-CATH polyclonal antibody and rat anti-CD68 monoclonal antibodyfollowed with fluorophore-conjugated secondary antibodies. The sectionswere imaged by confocal fluorescence microscopy. CATH and CD68-enrichedmacrophages labeling are shown separately in the indicated columns aswell as in the overlay image. All panels were also labeled to detectnuclei (DAPI-labeled) and panels A-I were additionally labeled for actinfilaments. Parenchymal tissues and surrounding regions of LG arepresented in panels A-I and while infiltrating foci are magnified inpanels J-L. Arrowheads point to CATH and CD68 double positive cells inthe surrounding region of the LG (C, F and I); arrows to the doublepositive cells in the interior region of the LG (C, F, and I) or in theinfiltrating foci (L). Panels a-i share the same magnification and themagnification bar for these figures is 20 μm, as shown in I. Panels J-Lshare the same magnification and the magnification bar for these figuresis 10 μm, as shown in L.

FIGS. 7A and B illustrate the comparison of CATH abundance and activityin LG lysates from 12-week old NOD and BALB/c mice. A: Western blottingto compare the protein abundance. 100 μg of lysate prepared from NODmouse LG (lanes 1 and 4), 100 μg of lysate prepared from BALB/c mouse LG(lanes 2 and 5) and 30 μg of Raw264.7 cell lysate (lanes 3 and 6) wereloaded onto an SDS-polyacrylamide gel. Upper panel: The proteinstransferred to nitrocellulose membranes were blotted with rat anti-CATHmonoclonal antibody with (1°+2°, lanes 1-3) or without (2°, lanes 4-6)primary antibody, then with IRDye 800-conjugated secondary antibody(lanes 1-6). Lower panel: The same membrane after being stripped wasre-blotted with rabbit anti-Rab3D polyclonal antibody and secondaryantibody (1°+2°, lanes 1-6) as a loading control. Major CATH bands aremarked by arrows and correspond to the single chain of one of the activeforms at 27-28 kD and the heavy chain of the other active form at 23-24kD (co-migrating with the positive control), as well as a possiblyproteolyzed or truncated species at 20 kD. These bands are most abundantin NOD mouse LG lysate. A band above the specific 23-24 kD band in lanes1-3 is visible due to non-specific reactivity of all samples with thegoat anti-rat secondary antibody (see lanes 4-6). The molecular weightsmarked indicate the migration of the molecular weight standards. B: CATHactivity assay. 10 μg each of paired LG lysates (n=3) were incubatedwith substrate in the absence (−inhibitor) or presence (+inhibitor) ofinhibitor. Samples labeled “no lysate” are the reaction background.Accumulated fluorescence of products from LG lysates was measured at 2h. Activity is fluorescence units (FU) per 10 μg lysate. Errors are ±SEMand *, p<0.05.

FIG. 8A shows that CtsS immunofluorescence is increased in male NODmouse LG, particularly in large SV (arrows); actin, red; bar, 10 μm.FIG. 8B shows CtsS activity in LG lysates and tears from 12 wk miceshows a significant (p<0.05) increase in male NOD mice. Inhibitorco-incubations show most activity is inhibited, verifying it is CtsS.

FIG. 9 shows that Lcn-2 immunofluorescence is detected at increasedintensity at the basolateral membranes as well as adjacent to or insidelumenal regions in the 12 week male NOD mouse LG relative to age-matchedBALB/c mouse LG. Actin filaments, and nuclei. Bar, 10 μm.

FIG. 10 shows human tear proteins separated by SDS-PAGE after beingeluted into cathepsin S assay buffer from Schirmer's test strips andbeing processed to analyze cathepsin S. In FIG. 10A, human tear proteinswere separated by SDS-PAGE after being processed through cathepsin Sassay. Protein was run on a 1.5 mm thick 10% gel and stained withGelCode Blue Stain Reagent (Pierce). C=fresh tears, SjS=Sjogren'spatient, N=normal patients. LE=left eye, RE=right eye. Molecular weightmarkers are in kDa. Max protein was loaded per lane, so amounts arevarying as labeled. In FIG. 10B, fresh human tears (33 mg, each lane)were separated by SDS-PAGE on a 0.75 mm thick 10% gel and stained withGelCode Blue Stain Reagent (Pierce). Patient 2 was a SjS patient.Patients 1 and 3 were normal patients with no disease. LE=left eye,RE=right eye. Molecular weight markers are in kDa.

FIG. 11 is a bar chart showing differences of activity levels ofcathepsin S in human tears collected by modification of the Schirmer'stest protocol onto filter paper strips followed by elution andmeasurement of CatS activity and protein. Values vary between genders.RE: right eye. LE: left eye.

FIG. 12 is a distribution curve showing that two of the three patientswith the highest cathepsin S activities in a set of clinical samplesplotted by eye were diagnosed with Sjögren's syndrome or lupus(indicated by the arrow).

FIG. 13 is a distribution bar chart showing that patients with Sjögren'ssyndrome or lupus were among the patients with the highest cathepsin Sactivities.

FIG. 14 shows that compared to other patients, cathepsin S activitylevels in Sjögren's syndrome or lupus patients were much higher.

FIG. 15 shows that compared to patients with other diseases, cathepsin Sactivity levels in Sjögren's syndrome or lupus patients were muchhigher.

FIG. 16 shows in female patients, CATS activities are the highest inpatients with the lowest Schirmer's values.

FIG. 17 shows in male patients, CATS activities are the highest inpatients with the lowest Schirmer's values.

FIGS. 18-21 show in patient groups of different ranges of Schirmer'svalues, the CATS activities among female and male patients.

FIGS. 22-25 show in patient groups of different ranges of Schirmer'svalues, the CATS activity values of specific patients.

FIGS. 26-29 show the CATS activities of female or male patients indifferent age groups.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature for example in the followingpublications. See, e.g., Sambrook and Russell eds. MOLECULAR CLONING: ALABORATORY MANUAL, 3^(rd) edition (2001); the series CURRENT PROTOCOLSIN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds. (2007)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc., N.Y.); PCR 1: A PRACTICALAPPROACH (M. MacPherson et al. IRL Press at Oxford University Press(1991)); PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames andG. R. Taylor eds. (1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow andLane eds. (1999)); CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE(R. I. Freshney 5^(th) edition (2005)); OLIGONUCLEOTIDE SYNTHESIS (M. J.Gait ed. (1984)); Mullis et al. U.S. Pat. No. 4,683,195; NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. (1984)); NUCLEIC ACIDHYBRIDIZATION (M. L. M. Anderson (1999)); TRANSCRIPTION AND TRANSLATION(B. D. Hames & S. J. Higgins eds. (1984)); IMMOBILIZED CELLS AND ENZYMES(IRL Press (1986)); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING(1984); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J. H. Miller and M.P. Calos eds. (1987) Cold Spring Harbor Laboratory); GENE TRANSFER ANDEXPRESSION IN MAMMALIAN CELLS (S. C. Makrides ed. (2003)) IMMUNOCHEMICALMETHODS IN CELL AND MOLECULAR BIOLOGY (Mayer and Walker, eds., AcademicPress, London (1987)); WEIR′S HANDBOOK OF EXPERIMENTAL IMMUNOLOGY (L. A.Herzenberg et al. eds (1996)).

DEFINITIONS

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include singular and plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a singlecell as well as a plurality of cells, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this invention.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. The term “about” also includes the exactvalue “X” in addition to minor increments of “X” such as “X+0.1” or“X−0.1.” It also is to be understood, although not always explicitlystated, that the reagents described herein are merely exemplary and thatequivalents of such are known in the art.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above.

The term “antigen” is well understood in the art and includes substanceswhich are immunogenic. The Ctss is an example of an antigen.

A “native” or “natural” or “wild-type” antigen is a polypeptide, proteinor a fragment which contains an epitope and which has been isolated froma natural biological source. It also can specifically bind to an antigenreceptor.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule. Examples of such include,but are not limited to a complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, a heavy chainor light chain variable region, a heavy chain or light chain constantregion, a framework (FR) region, or any portion thereof, or at least oneportion of a binding protein, any of which can be incorporated into anantibody of the present invention.

In one aspect, the “biological activity” means the ability of theantibody to selectively bind its epitope protein or fragment thereof asmeasured by ELISA or other suitable methods. Biologically equivalentantibodies, include but are not limited to those antibodies, peptides,antibody fragments, antibody variant, antibody derivative and antibodymimetics that bind to the same epitope as the reference antibody.

The term “antibody” is further intended to encompass digestionfragments, specified portions, derivatives and variants thereof,including antibody mimetics or comprising portions of antibodies thatmimic the structure and/or function of an antibody or specified fragmentor portion thereof, including single chain antibodies and fragmentsthereof. Examples of binding fragments encompassed within the term“antigen binding portion” of an antibody include a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH, domains; aF(ab′)² fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; a Fd fragmentconsisting of the VH and CH, domains; a Fv fragment consisting of the VLand VH domains of a single arm of an antibody, a dAb fragment (Ward etal. (1989) Nature 341:544-546), which consists of a VH domain; and anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston etal. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. Single chainantibodies are also intended to be encompassed within the term “fragmentof an antibody.” Any of the above-noted antibody fragments are obtainedusing conventional techniques known to those of skill in the art, andthe fragments are screened for binding specificity and neutralizationactivity in the same manner as are intact antibodies.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “antibody variant” is intended to include antibodies producedin a species other than a mouse. It also includes antibodies containingpost-translational modifications to the linear polypeptide sequence ofthe antibody or fragment. It further encompasses fully human antibodies.

The term “antibody derivative” is intended to encompass molecules thatbind an epitope as defined above and which are modifications orderivatives of a native monoclonal antibody of this invention.Derivatives include, but are not limited to, for example, bispecific,multispecific, heterospecific, trispecific, tetraspecific, multispecificantibodies, diabodies, chimeric, recombinant and humanized.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1), C_(H2),C_(H3)), hinge, (VL, VH)) is substantially non-immunogenic in humans,with only minor sequence changes or variations. Similarly, antibodiesdesignated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse,rat, rabbit, guinea pig, hamster, and the like) and other mammalsdesignate such species, sub-genus, genus, sub-family, family specificantibodies. Further, chimeric antibodies include any combination of theabove. Such changes or variations optionally and preferably retain orreduce the immunogenicity in humans or other species relative tonon-modified antibodies. Thus, a human antibody is distinct from achimeric or humanized antibody. It is pointed out that a human antibodycan be produced by a non-human animal or prokaryotic or eukaryotic cellthat is capable of expressing functionally rearranged humanimmunoglobulin (e.g., heavy chain and/or light chain) genes. Further,when a human antibody is a single chain antibody, it can comprise alinker peptide that is not found in native human antibodies. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

A hybridoma is a cell that is produced in the laboratory from the fusionof an antibody-producing lymphocyte and a non-antibody producing cancercell, usually a myeloma or lymphoma. A hydridoma proliferates andproduces a continuous supply of a specific monoclonal antibody.

A “human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable and constant regions derivedfrom human germline immunoglobulin sequences.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

An “autoimmune disease” intends a disease that arises from an overactiveimmune response of the body against substances and tissues normallypresent in the body in which the body actually attacks its own cells.Examples of autoimmune diseases include, without limitation, Coeliacdisease, diabetes mellitus type 1 (IDDM), lupus erythematosus, systemiclupus erythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome,Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenicpurpura, rheumatoid arthritis (RA), ankylosing spondylitis, Crohnsdisease, dermatomyositis, Goodpasture's syndrome, Guillain-Barrésyndrome (GBS), mixed Connective tissue disease, multiple sclerosis,myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anaemia,psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis,relapsing polychondritis, temporal arteritis, ulcerative colitis,vasculitis and Wegener's granulomatosis.

“Ctss” or “cathepsin S” is member of the peptidase C1 family, alysosomal cysteine proteinase that may participate in the degradation ofantigenic proteins to peptides for presentation on MHC class IImolecules. The encoded protein can function as an elastase over a broadpH range in alveolar macrophages. Representative sequences includeUniProtKB: P25774 and Entrez Gene: 1520.

“Ctsh” or “cathepsin H” is a lysosomal cysteine proteinase important inthe overall degradation of lysosomal proteins. It is composed of a dimerof disulfide-linked heavy and light chains, both produced from a singleprotein precursor. The encoded protein, which belongs to the peptidaseC1 protein family, can act both as an aminopeptidase and as anendopeptidase. Increased expression of this gene has been correlatedwith malignant progression of prostate tumors. Representative sequencesinclude UniProtKB: P09668 and Entrez Gene: 1512.

“Ctsr” or “cathepsin R” is a lysosomal cysteine proteinase and member ofthe peptidase C1 family. Ctsr was identified as a candidate lung tumorsusceptibility gene identified through whole-genome association analysesin inbred mice. Representative sequences include UniProKB: □9JLA9 andGeneBank Protein ID: NP_(—)064680.

“Ctsw” or “cathepsin W”, a member of the peptidase C1 family, is acysteine proteinase that may have a specific function in the mechanismor regulation of T-cell cytolytic activity. The encoded protein is foundassociated with the membrane inside the endoplasmic reticulum of naturalkiller and cytotoxic T-cells. Expression of this gene is up-regulated byinterleukin-2 Representative sequences include UniProtKB: P56202 andEntrez Gene: 1521.

“Ctsz” or “cathepsin Z” is a lysosomal cysteine proteinase and member ofthe peptidase C1 family. It exhibits both carboxy-monopeptidase andcarboxy-dipeptidase activities. The encoded protein has also been knownas cathepsin X and cathepsin P. This gene is expressed ubiquitously incancer cell lines and primary tumors and, like other members of thisfamily, may be involved in tumorigenesis. Representative sequencesinclude UniProtKB: Q9UBR2 and Entrez Gene: 1522.

“Ifng” or “interferon, gamma” is a cytokine critical for innate andadaptive immunity against viral and intracellular bacterial infectionsand for tumor control. Aberrant IFNG expression is associated with anumber of autoinflammatory and autoimmune diseases. The importance ofIFNG in the immune system stems in part from its ability to inhibitviral replication directly, but most importantly derives from itsimmunostimulatory and immunomodulatory effects. IFNG is producedpredominantly by natural killer (NK) and natural killer T (NKT) cells aspart of the innate immune response, and by CD4 and CD8 cytotoxic Tlymphocyte (CTL) effector T cells once antigen-specific immunitydevelops. Representative sequences include UniProtKB: P01579 and EntrezGene: 3458.

Il6ra″ or “interleukin 6 receptor alpha subunit” is a potent pleiotropiccytokine that regulates cell growth and differentiation and plays animportant role in immune response. The protein encoded by this gene is asubunit of the receptor complex for IL6. The IL6 receptor is a proteincomplex consisting of this protein and interleukin 6 signal transducer(IL6ST/GP130/IL6-beta), a receptor subunit also shared by many othercytokines Dysregulated production of IL6 and this receptor areimplicated in the pathogenesis of many diseases, such as multiplemyeloma, autoimmune diseases and prostate cancer. Alternatively splicedtranscript variants encoding distinct isoforms have been reported.Representative sequences include UniProtKB: P08887 and Entrez Gene:3570.

“Il10” or “interleukin 10” is a cytokine produced primarily by monocytesand to a lesser extent by lymphocytes. This cytokine has pleiotropiceffects in immunoregulation and inflammation. It down-regulates theexpression of Th1 cytokines, MHC class II Ags, and costimulatorymolecules on macrophages. It also enhances B cell survival,proliferation, and antibody production. This cytokine can block NF-kappaB activity, and is involved in the regulation of the JAK-STAT signalingpathway. Knockout studies in mice suggested the function of thiscytokine as an essential immunoregulator in the intestinal tract.Representative sequences include UniProtKB: P22301 and Entrez Gene:3586.

“Il10ra” or “interleukin 10 receptor, alpha” is a receptor forinterleukin 10. This protein is structurally related to interferonreceptors. It has been shown to mediate the immunosuppressive signal ofinterleukin 10, and thus inhibits the synthesis of proinflammatorycytokines. This receptor is reported to promote survival of progenitormyeloid cells through the insulin receptor substrate-2/PI 3-kinase/AKTpathway. Activation of this receptor leads to tyrosine phosphorylationof JAK1 and TYK2 kinases. Representative sequences include UniProtKB:Q13651 and Entrez Gene: 3587.

Il15″ or “interleukin 15” is a cytokine that regulates T and naturalkiller cell activation and proliferation. This cytokine and interleukin2 share many biological activities. They are found to bind commonhematopoietin receptor subunits, and may compete for the same receptor,and thus negatively regulate each other&apos;s activity. The number ofCD8+ memory cells is shown to be controlled by a balance between thiscytokine and IL2. This cytokine induces the activation of JAK kinases,as well as the phosphorylation and activation of transcriptionactivators STAT3, STAT5, and STAT6. Studies of the mouse counterpartsuggested that this cytokine may increase the expression of apoptosisinhibitor BCL2L1/BCL-x(L), possibly through the transcription activationactivity of STAT6, and thus prevent apoptosis. Two alternatively splicedtranscript variants of this gene encoding the same protein have beenreported. Representative sequences include UniProtKB: P40933 and EntrezGene: 3600.

“TNFα or Tnfa” or “tumor necrosis factor alpha” is a multifunctionalproinflammatory cytokine that belongs to the tumor necrosis factor (TNF)superfamily. This cytokine is mainly secreted by macrophages. It canbind to, and thus functions through its receptors TNFRSF1A/TNFR1 andTNFRSF1B/TNFBR. This cytokine is involved in the regulation of a widespectrum of biological processes including cell proliferation,differentiation, apoptosis, lipid metabolism, and coagulation. Thiscytokine has been implicated in a variety of diseases, includingautoimmune diseases, insulin resistance, and cancer. Knockout studies inmice also suggested the neuroprotective function of this cytokineRepresentative sequences include UniProtKB: P01375 and Entrez Gene:7124.

“Apo-F” or “apolipoprotein F” is one of the minor apolipoproteins foundin plasma. This protein forms complexes with lipoproteins and may beinvolved in transport and/or esterification of cholesterol.Representative sequences include UniProtKB: Q13790 and Entrez Gene: 319.

“Lcn-2”, “lipocalin 2”, or “neutrophil gelatinase-associated lipocalin”,is expressed in both mice and humans, and has been shown to be highlyexpressed in pancreatic islets, bone marrow, and SG. Until now itsexpression in LG has not been investigated but microarray data suggestmoderate to high levels of expression. It is implicated in diversebioprocesses including iron-siderophore binding in bacterial infectionsas a component of the innate immune system, modulation of inflammation,and is a marker closely related to obesity and insulin resistance (Flo,et al. (2004) Nature 432(7019):917-21, 13). Lcn-2 transports smalllipophilic substances. Representative sequences include UniProtKB:P80188 and Entrez Gene: 3934.

“Lactoperoxidase”, “Salivary peroxidase” or “LPO” has representativesequences including UniProtKB: P22079 and Entrez Gene: 4025.

“Lactoferrin” or “LTF” is a member of the transferrin family of genesand its protein product is found in the secondary granules ofneutrophils. The protein is a major iron-binding protein in milk andbody secretions with an antimicrobial activity, making it an importantcomponent of the non-specific immune system. The protein demonstrates abroad spectrum of properties, including regulation of iron homeostasis,host defense against a broad range of microbial infections,anti-inflammatory activity, regulation of cellular growth anddifferentiation and protection against cancer development andmetastasis. Representative sequences include UniProtKB: P02788 andEntrez Gene: 4057.

“Lysozyme” or “LYZ” has a natural substrate that is the bacterial cellwall peptidoglycan (cleaving the beta[1-4]glycosidic linkages betweenN-acetylmuramic acid and N-acetylglucosamine). Lysozyme is one of theanti-microbial agents found in human milk, and is also present inspleen, lung, kidney, white blood cells, plasma, saliva, and tears.Missense mutations in LYZ have been identified in heritable renalamyloidosis. Representative sequences include UniProtKB: P61626 andEntrez Gene: 4069.

The term “recombinant protein” refers to a polypeptide which is producedby recombinant DNA techniques, wherein generally, DNA encoding thepolypeptide is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

A “mammal” is a class of vertebrate animals whose females arecharacterized by the possession of mammary glands while both males andfemales are characterized by sweat glands, hair, three middle ear bonesused in hearing, and a neocortex region in the brain. Non-limitingexamples of a mammal include a simian, a murine, a bovine, an equine, aporcine or an ovine. In one aspect, a mammal is a mouse. In anotheraspect, a mammal is a rat. In yet another aspect, a mammal is a rabbit.In yet another aspect, a mammal is a human.

“Expression” as applied to a gene or a protein, refers to the productionof the mRNA transcribed from the gene or the protein product encoded bythe gene. In one aspect, “expression” level is determined by measuringthe expression level of a gene of interest for a given patientpopulation, determining the median expression level of that gene for thepopulation, and comparing the expression level of the same gene for asingle patient to the median expression level for the given patientpopulation. For example, if the expression level of a gene of interestfor the single patient is determined to be above the median expressionlevel of the patient population, that patient is determined to have highexpression of the gene of interest. Alternatively, if the expressionlevel of a gene of interest for the single patient is determined to bebelow the median expression level of the patient population, thatpatient is determined to have low expression of the gene of interest.

“Overexpression” or “underexpression” refers to increased or decreasedexpression, or alternatively a differential expression, of a gene in atest sample as compared to the expression level of that gene in thecontrol sample. In one aspect, the test sample is a diseased cell, andthe control sample is a normal cell. In another aspect, the test sampleis an experimentally manipulated or biologically altered cell, and thecontrol sample is the cell prior to the experimental manipulation orbiological alteration. In yet another aspect, the test sample is asample from a patient, and the control sample is a similar sample from ahealthy individual. In a yet further aspect, the test sample is a samplefrom a patient and the control sample is a similar sample from patientnot having the desired clinical outcome. In one aspect, the differentialexpression is about 1.5 times, or alternatively, about 2.0 times, oralternatively, about 2.0 times, or alternatively, about 3.0 times, oralternatively, about 5 times, or alternatively, about 10 times, oralternatively about 50 times, or yet further alternatively more thanabout 100 times higher or lower than the expression level detected inthe control sample. Alternatively, the gene is referred to as “overexpressed” or “under expressed”. Alternatively, the gene may also bereferred to as “up regulated” or “down regulated”.

An “internal control” or “house keeping” gene refers to anyconstitutively or globally expressed gene whose presence enables anassessment of the gene of interests expression level. Such an assessmentcomprises a determination of the overall constitutive level of genetranscription and a control for variation in sampling error. Examples ofsuch genes include, but are not limited to, β-actin, the transferrinreceptor gene, GAPDH gene or equivalents thereof. In some aspects ofthis invention, the internal control or house keeping gene is thesuitable control.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The phrase “amplification of polynucleotides” includes methods such asPCR, ligation amplification (or ligase chain reaction, LCR) andamplification methods. These methods are known and widely practiced inthe art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis etal., 1990 (for PCR); and Wu, D. Y. et al. (1989) Genomics 4:560-569 (forLCR). In general, the PCR procedure describes a method of geneamplification which is comprised of (i) sequence-specific hybridizationof primers to specific genes within a DNA sample (or library), (ii)subsequent amplification involving multiple rounds of annealing,elongation, and denaturation using a DNA polymerase, and (iii) screeningthe PCR products for a band of the correct size. The primers used areoligonucleotides of sufficient length and appropriate sequence toprovide initiation of polymerization, i.e. each primer is specificallydesigned to be complementary to each strand of the genomic locus to beamplified.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid molecule comprising an open reading frame and including atleast one exon and (optionally) an intron sequence. The term “intron”refers to a DNA sequence present in a given gene which is spliced outduring mRNA maturation.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present invention.

The term “interact” as used herein is meant to include detectableinteractions between molecules, such as can be detected using, forexample, a hybridization assay. The term interact is also meant toinclude “binding” interactions between molecules. Interactions may be,for example, protein-protein, protein-nucleic acid, protein-smallmolecule or small molecule-nucleic acid in nature.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term “isolated” is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,derivatives, variants and analogs of either RNA or DNA made fromnucleotide analogs, and, as applicable to the embodiment beingdescribed, single (sense or antisense) and double-strandedpolynucleotides. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes ofclarity, when referring herein to a nucleotide of a nucleic acid, whichcan be DNA or an RNA, the terms “adenosine”, “cytidine”, “guanosine”,and “thymidine” are used. It is understood that if the nucleic acid isRNA, a nucleotide having a uracil base is uridine.

The terms “oligonucleotide” or “polynucleotide”, or “portion,” or“segment” thereof refer to a stretch of polynucleotide residues which islong enough to use in PCR or various hybridization procedures toidentify or amplify identical or related parts of mRNA or DNA molecules.The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

When gene or protein expression level “is used as a basis” for selectinga patient for a treatment described herein, the gene or proteinexpression level is measured before and/or during treatment, and thevalues obtained are used by a clinician in assessing any of thefollowing: (a) probable or likely suitability of an individual toinitially receive treatment(s); (b) responsiveness to treatment; (c)probable or likely suitability of an individual to continue to receivetreatment(s); (d) adjusting dosage; (e) predicting likelihood ofclinical benefits. As would be well understood by one in the art,measurement of the gene expression level in a clinical setting is aclear indication that this parameter was used as a basis for initiating,continuing, adjusting and/or ceasing administration of the treatmentsdescribed herein.

The term “treating” as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.

“An effective amount” intends to indicate the amount of a compound oragent administered or delivered to the patient which is most likely toresult in the desired treatment outcome. The amount is empiricallydetermined by the patient's clinical parameters including, but notlimited to the stage of disease, age, gender, histology, and likelihoodfor recurrence.

“Sjögren's syndrome” or “SjS” affects an estimated 4 million Americans,with 9 out of 10 patients being women (Lemp (2005) Am J Ophthalmol.140(5):898-9; Hansen., et al. (2005) Curr Opin Rheumatol. 17(5):558-65).SjS is the second most common autoimmune disease in the United States;research into the improved diagnosis and treatment for these autoimmunedisorders has long been recognized by the Office for Research on Women'sHealth (ORWH) as a priority. The lacrimal gland (LG) is responsible forsecretion of proteins and fluid to sustain the health of the ocularsurface (OS). SjS is characterized by lymphocytic infiltration of LG andsalivary gland (SG), followed by development of functional quiescence(e.g., inability to secrete fluid and proteins) as well as the eventualinflammatory destruction of these glands. The ocular surface of SjSpatients becomes desiccated and prone to infection because of thefunctional quiescence of the gland as well as changes in the spectrum ofsecreted proteins, leading to severe corneal damage and in some cases,blindness. SjS can occur independently (primary Sj S) or in conjunctionwith another autoimmune disease such as rheumatoid arthritis or systemiclupus erythematosus (secondary SjS). Primary SjS in particular isassociated with significant effects on other organs including the brain,kidneys, lungs, pancreas and gastrointestinal tract (Hansen., et al.(2005) Curr Opin Rheumatol. 17(5):558-65)). A diagnosis of primary SjSis also associated with a significantly increased risk of B celllymphoma. In primary and secondary SjS, the presenting symptoms overlapwith other keratoconjunctivitis sicca (KCS) disorders while forsecondary SjS, the presenting symptoms also overlap with many otherautoimmune diseases. The SjS Foundation estimates that it requiresnearly 7 years for the typical patient to receive a diagnosis of SjS,because the symptoms overlap so substantially with those for otherdiseases. Since the tears and saliva are thought to be uniquely alteredin SjS, it is astounding that there are currently no tear or salivarybiomarkers that are recognized as diagnostic for SjS, in particular forprimary SjS, and which can aid in the early identification of thissubset of SjS patients that will experience more severe disease.Achievement of the goals proposed in this application, detection andvalidation of unique tear biomarkers that are released into tears inproportion to the severity of the disease and development of newparadigms for assay of such materials in tear fluid, would be criticalfirst steps in the ultimate goal of identification and earlier treatmentof SjS patients to arrest aggressive disease development. Since SjS,like most autoimmune diseases, is so strongly manifested inpost-menopausal women relative to other populations, the success of thisinvention will also constitute a critical advance in diagnosis of adisease that has a tremendous impact on women's health.

Disease models for SjS include the NOD mouse, a genetic model and theIL-1-injected BALB/c mouse. They represent two models of inflammatoryautoimmune LG disease. These two models differ in that one is a geneticmodel and the other is an experimentally-induced disease model whichuses cytokine injection to induce LG disease.

“NOD mouse” is a well-studied genetic animal model for humaninsulin-dependent diabetes mellitus and SjS (Barabino, et al. (2004)Invest Ophthalmol V is Sci. 45(6):1641-6). This strain spontaneouslydevelops lymphocytic infiltration in submandibular glands(sialoadenitis) and LG (dacryoadenitis), and diabetes. Male NOD mice aresignificantly more susceptible to dacryoadenitis, and diseasedevelopment in the female mouse LG is minimal. This differs from thehuman disease since SjS is more prevalent in women. Despite thisdifference, the lymphocytic infiltration into the LG of male mice isprofound and occurs as early as 6 weeks, along with decreased productionof tear fluid. The male NOD mouse LG also exhibits significant lipiddeposition, a feature of human SjS. This genetic model system has beenuseful in generating strong leads for the protein biomarkers in tearfluid that are proposed to increase in disease: CtsS, Apo-F and Lcn-2.Male NOD mice, which develop the severe autoimmune inflammatory LGdisease, will be utilized for tear fluid collection, in parallel withmeasurement of tear flow and corneal, conjunctival and LG integrity andinflammation. The ages of mice examined can span disease onset (4-12weeks), intermediate development of disease (12 weeks-6 months) andadvanced disease (6-12 months). Age- and gender-matched BALB/c mice canbe used as controls.

“IL-1-injected BALB/c mouse” is a mouse model of induced inflammatoryautoimmune disease that will be utilized as a second model is based on asingle injection of IL-1β or IL-1α into LG of female BALB/c or C57BL/6mice, which induces a reversible inflammatory aqueous tear deficiency(Zoukhri, et al. (2002) Invest Ophthalmol V is Sci. 43(5):1429-36),followed by recovery within a defined timetable which is dependent uponthe injected strain.

Schirmer's test strips have traditionally been used for auantitativemeasurement of tear production. The standardized Schirmer test stripconsists of a 5×35 mm strip of Whatman #41 filter paper; the paper has anotch located 5 mm from one end of the strip. The strips arecommercially available from vendors such as Alcon Manufacturing, Ltd.The notched end of the strip can be is rounded. As described in U.S.Pat. No. 5,006,310 a Schirmer Tear Test is performed by bending thestrip at the notch (˜120° bend). The rounded end of the Schirmer TearTest strip is then inserted into the lower conjunctival sac of each eye.The eyes are then closed and the strip is progressively wetted bycapillary action drawing up tears as they are produced. The distance thetear migration front has moved is measured after 5 minutes. Themigration distance of the tears is measured from the notch of the stripas the zero point. Reading the test involves removing the strip from theeye and placing it against a scale graduated in millimeters. 15 mm ofwetting in 5 minutes is considered normal. For tear production levels,it has been recommended that the tear migration front be measured asclose to the 5 minute time mark as possible because the tear front willcontinue to migrate up the strip after the strip is removed from theeye. Thus, late readings give rise to results that are artificiallyhigh.

As used herein, the term “detectable label” intends a directly orindirectly detectable compound or composition that is conjugateddirectly or indirectly to the composition to be detected, e.g.,polynucleotide or protein such as an antibody so as to generate a“labeled” composition. The term also includes sequences conjugated tothe polynucleotide that will provide a signal upon expression of theinserted sequences, such as green fluorescent protein (GFP) and thelike. The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. The labels can be suitable for small scale detection or moresuitable for high-throughput screening. As such, suitable labelsinclude, but are not limited to radioisotopes, fluorochromes,chemiluminescent compounds, dyes, and proteins, including enzymes. Thelabel may be simply detected or it may be quantified. A response that issimply detected generally comprises a response whose existence merely isconfirmed, whereas a response that is quantified generally comprises aresponse having a quantifiable (e.g., numerically reportable) value suchas an intensity, polarization, and/or other property. In luminescence orfluoresecence assays, the detectable response may be generated directlyusing a luminophore or fluorophore associated with an assay componentactually involved in binding, or indirectly using a luminophore orfluorophore associated with another (e.g., reporter or indicator)component.

Examples of luminescent labels that produce signals include, but are notlimited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed for example in Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Examples of suitable fluorometric labels include, but are not limitedto, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

Descriptive Embodiments

It has been discovered herein that the expression level of activitycertain peptides are altered in tears in patients having an autoimmunedisease. The altered expression level or activity of these polypeptidesthen can be used as biomarker for diagnosis or prediction of autoimmunediseases, methods of restoring the expression level or activity,therefore, can be used to treat the autoimmune disease.

Thus, the invention, in one aspect, provides diagnostic methods, whichare based, at least in part, on determination of the expression oractivity levels of a polypeptide selected from the group Ctss, Ctsh,Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10, Il10ra, Il15, Tnfa, Apo-F, Lcn-2,lactoperoxidase, lactoferrin or lysozyme. In one aspect at least onepolypeptide expression level is determined, in another at least two, oralternatively at least three, or alternatively at least 4, oralternatively at least 5, or alternatively at least 6, or alternativelyat least 7, or alternatively at least 8, or alternatively at least 9, oralternatively at least 10, or alternatively at least 11, oralternatively at least 12, or alternatively at least 13, oralternatively at least 14, or alternatively at least 15, oralternatively all 16 expression levels are determined. In a furtheraspect, measuring the expression level or activity level of Apo-F isexcluded from the method.

In one aspect, the expression or activity levels of a polypeptideselected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10,Il10ra, Il15, Tnfa, Apo-F, Lcn-2, lactoperoxidase, lactoferrin orlysozyme is used as a basis for selecting a patient for a treatment. Inone aspect at least one polypeptide expression level is determined, inanother at least two, or alternatively at least three, or alternativelyat least 4, or alternatively at least 5, or alternatively at least 6, oralternatively at least 7, or alternatively at least 8, or alternativelyat least 9, or alternatively at least 10, or alternatively at least 11,or alternatively at least 12, or alternatively at least 13, oralternatively at least 14, or alternatively at least 15, oralternatively all 16 expression levels are determined. In a furtheraspect, measuring the expression level or activity level of Apo-F isexcluded from the method.

In one embodiment, the invention provides a method for determiningwhether a mammal is likely to develop Sjögren's syndrome, comprising, oralternatively consisting essentially of or yet further consisting ofmeasuring an expression level or activity level of at least one firstpolypeptide selected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng,Il6ra, Il10, Il10ra, Il15, Tnfa, Apo-F, or Lcn-2, and/or at least onesecond polypeptide selected from the group lactoperoxidase, lactoferrinor lysozyme in a sample of tear isolated from the mammal, wherein anincreased expression level or increased activity level of the firstpolypeptide or a decreased expression level or decreased activity levelof the second polypeptide, as compared to a suitable control, indicatesthat the mammal is likely to develop autoimmune disease. In one aspectat least one polypeptide expression level of a first polypeptide isdetermined, in another at least two, or alternatively at least three, oralternatively at least 4, or alternatively at least 5, or alternativelyat least 6, or alternatively at least 7, or alternatively at least 8, oralternatively at least 9, or alternatively at least 10, or alternativelyat least 11, or alternatively at least 12, or alternatively all 13expression levels are determined. In one aspect at least one polypeptideexpression level of a second polypeptide is determined, in another atleast two, or alternatively all three of the second polypeptidesexpression levels are determined. In a further aspect, measuring theexpression level or activity level of Apo-F is excluded from the method.

Methods of measuring the expression level of polypeptide are known inthe art. Non-limiting examples include Western blot, gelelectrophoresis, ELISA, fluorometric analysis mass spectrometry, orprotein array.

“Activity level” as applied to a protein, refers to the enzymaticactivity level of the protein. Determination of the activity level canbe made based on the capability of the protein to catalyze a chemical orbiological reaction using one or more substrates. In one aspect,activity level is protease activity level which can be determined by theprotein's capability to hydrolyze a peptide sequence at a specificlocation.

Methods of measuring the activity level of a polypeptide are known inthe art. For example, for polypeptides that have protease activities,their activities can be measured by their capability to hydrolyze asubstrate peptide. Protease activity measuring kits for variousproteases including Ctss are commercially available from vendors such asSigma-Aldrich (St. Louis, Mo.) and BioVision Inc. (Mountain View,Calif.).

Measurement of protein expression level or activity level can be made incomparison to suitable controls. Suitable internal controls can be aprotein or other agent that is constantly present in the same samplefrom different mammals. Suitable internal controls can also be the totalvolume of samples collected, such as the total volume of tear fluid.Suitable external controls can also used for determination of theprotein expression level or activity level. Suitable external controlscan be a mammal that does not appear to have the disease of interest.Suitable external controls can also be historical samples collected thathave been proven to be from mammals that do not have the disease. In oneaspect, a suitable external control for a NOD mouse is a BALB/c mouse.

Collection of samples of tear can be done with methods known in the artand described briefly herein. Stimulation can be applied when needed,for example by eye-wash prior to sample collection. For the purpose ofillustration only, tears can be collected onto a Schirmer's test stripcontaining or embedded with a quantitatively-labeled substrate, e.g., anantibody coupled to a detectable marker such as a fluorometric label orsubstrate. The polypeptide or protein in the tear will react with thedetectably labeled substrate allowing measurement of the expressionlevel or activity level of the one first and/or second polypeptide.

As used herein, a mammal that is “likely to develop autoimmune disease”is a mammal that is more likely than not to develop autoimmune disease.

In another embodiment, the invention provides a method for aiding indiagnosing autoimmune disease in a mammal, comprising, or alternativelyconsisting essentially of or yet further consisting of measuring anexpression level or activity level of at least one first polypeptideselected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10,Il10ra, Il15, Tnfa, Apo-F, or Lcn-2, and/or at least one secondpolypeptide selected from the group lactoperoxidase, lactoferrin orlysozyme in a sample of tear isolated from the mammal, wherein anincreased expression level or increased activity level of the firstpolypeptide or a decreased expression level or decreased activity levelof the second polypeptide, as compared to a suitable control, indicatesa likely positive diagnosis of autoimmune disease for the mammal,thereby aiding in the diagnosis. In one aspect at least one polypeptideexpression level of a first polypeptide is determined, in another atleast two, or alternatively at least three, or alternatively at least 4,or alternatively at least 5, or alternatively at least 6, oralternatively at least 7, or alternatively at least 8, or alternativelyat least 9, or alternatively at least 10, or alternatively at least 11,or alternatively at least 12, or alternatively all 13 expression levelsare determined. In one aspect at least one polypeptide expression levelof a second polypeptide is determined, in another at least two, oralternatively all three of the second polypeptides expression levels aredetermined. In a further aspect, measuring the expression level oractivity level of Apo-F is excluded from the method.

In one aspect, aiding in the diagnosis refers to providing confirmationto existing diagnosis. In another aspect, aiding in the diagnosis refersto using the diagnosis method in a panel of diagnosis methods, eachmethod of the panel contributing to a final diagnosis. In yet anotheraspect, aiding in the diagnosis refers to that more than one of themarkers recited herein are used in combination to make a diagnosis.

In another embodiment, the invention provides a method for diagnosingrelative severity of autoimmune disease in a mammal, comprising, oralternatively consisting essentially of or yet further consisting ofmeasuring an expression level or activity level of a first polypeptideselected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10,Il10ra, Il15, Tnfa, Apo-F, or Lcn-2, or a second polypeptide selectedfrom the group lactoperoxidase, lactoferrin or lysozyme in a sample oftear from the mammal, wherein a relatively higher expression level oractivity level of the first polypeptide or a relatively lower expressionlevel or activity level of the second polypeptide, as compared to asuitable control, indicates that the individual has relatively moresevere autoimmune disease. In one aspect at least one polypeptideexpression level of a first polypeptide is determined, in another atleast two, or alternatively at least three, or alternatively at least 4,or alternatively at least 5, or alternatively at least 6, oralternatively at least 7, or alternatively at least 8, or alternativelyat least 9, or alternatively at least 10, or alternatively at least 11,or alternatively at least 12, or alternatively all 13 expression levelsare determined. In one aspect at least one polypeptide expression levelof a second polypeptide is determined, in another at least two, oralternatively all three of the second polypeptides expression levels aredetermined. In a further aspect, measuring the expression level oractivity level of Apo-F is excluded from the method.

It is to be intended, although not always explicitly stated, that themethods of this invention can be further modified by measuring ordetermining the expression level or activity level of at least two, oralternatively at least three, or alternatively at least four, oralternatively at least five, or alternatively at least six, oralternatively at least seven, or alternatively at least eight of thepolypeptides are measured and compared to suitable controls, and adiagnosis can be made based on their overall expression level oractivity level changes.

In one aspect of the above embodiments, the first polypeptide isselected from the group Ctss, Apo-F or Lcn-2. In a further aspect,measuring the expression level or activity level of Apo-F is excludedfrom the method.

Further, it has now been discovered that, similar to a mouse, humanpatients in which Apo-F is dispersedly distributed within cytoplasmicand secretory vesicle-like organelles may likely be suffering fromautoimmune disease or at risk of developing autoimmune disease, whereashuman individuals in which Apo-F is located at the cytoplasmic face ofthe plasma membrane in the acinar cells are likely not suffering fromautoimmune disease or at risk of developing autoimmune disease. Thedispersed distribution of ApoF in NOD mouse LG and certain human LG isbelieved to result from missorting of ApoF in the acinar cells.

Without being bound by theory, Applicants' finding indicates that thedisparate distribution patterns of missorted proteins, such as Apo-F aswell as CATS, as observed in the NOD mouse model compared to healthymice, are markers for the diagnosis of autoimmune disease, not only inmouse, but also in human and other mammals. Accordingly, the presence ofApo-F or CATS at the cytoplasmic face of the plasma membrane in acinarcells in a LG from a mammal indicates that the mammal is not likelysuffering from autoimmune disease or is not at risk of developingautoimmune disease. A dispersed distribution of Apo-F or CATS withincytoplasmic or secretory vesicle-like organelles in acinar cells in a LGfrom a mammal indicates that the mammal is suffering from autoimmunedisease or is at risk of developing autoimmune disease.

Accordingly, one aspect of the invention provides a method fordetermining whether a mammal is likely to develop autoimmune disease,comprising determining the presence of a protein selected from CATS orAPO-F or any missorted protein as observed in the NOD mouse LG, in anacinar cell isolated from the lacrimal gland or salivary gland of themammal, wherein the mammal is likely to develop autoimmune disease ifmore than about 10%, or alternatively about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% ofthe protein in the cell is present not in proximity to the basolateralmembrane of the cell. As is apparent to those skilled in the art, afailure of this finding indicates that the mammal is not likely todevelop autoimmune disease.

Further provided is a method for aiding in diagnosing autoimmune diseasein a mammal, comprising determining the presence of a protein selectedfrom CATS or APO-F or any missorted protein as observed in the NOD mouseLG, in an acinar cell isolated from the lacrimal gland or salivary glandof the mammal, wherein presence of more than about 10%, or alternativelyabout 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90%, or about 95% of the protein in the cell not in proximityto the basolateral membrane of the cell indicates a likely positivediagnosis of autoimmune disease for the mammal, thereby aiding in thediagnosis. As is apparent to those skilled in the art, a failure of thisfinding indicates that the mammal is not a positive diagnosis to developautoimmune disease.

As used in herein, the term “proximity” intends a distance in a cellthat is relative small compared to the diameter of the cell. In oneaspect, not in proximity to the basolateral membrane of the cell meanshaving a distance from the basolateral membrane of the cell that is atleast about 1/20, or alternatively about 1/10, about 1/9, about ⅛, about1/7, about ⅙, about ⅕, about ¼, or about ⅓ of the diameter of the cell.

In another aspect of the above embodiments, the method further comprisesdiagnosing the mammal with a test selected from the group of Schirmertest, a slit-lamp examination, a radiological test, or a blood test. Theresults from this additional test can be combined with the methodsprovided in the above embodiment to assist diagnosis. In this aspect,the methods as disclosed herein aid in the diagnosis of autoimmunedisease when combined with other known or yet to be developed diagnosticmethods.

Schirmer's test determines whether the eye produces enough tears to keepit moist. This test is used when a person experiences very dry eyes orexcessive watering of the eyes. Schirmer's test uses paper stripsinserted into the eye for several minutes to measure the production oftears. This technique measures basic tear function. Applicants haveadapted these filter paper strips in common usage to collect human tearsfor measurement of protein activities.

A slit-lamp examination uses an instrument, slit-lamp, to provide amagnified, three-dimensional view of the different parts of the eye. Theslit lamp is an instrument consisting of a high-intensity light sourcethat can be focused to shine a thin sheet of light into the eye. It isused in conjunction with a biomicroscope. The lamp facilitates anexamination of the anterior segment, or frontal structures and posteriorsegment, of the human eye, which includes the eyelid, sclera,conjunctiva, iris, natural crystalline lens, and cornea. The binocularslit-lamp examination provides stereoscopic magnified view of the eyestructures in detail, enabling anatomical diagnoses to be made for avariety of eye conditions.

A radiological procedure can also be used as a reliable and accurate wayof diagnosing autoimmune disease. A contrast agent is injected into theparotid duct (of Stensen), which is a duct opening from the cheek intothe vestibule of the mouth opposite the neck of the upper second molartooth. Widespread puddling of the injected contrast scattered throughoutthe gland indicates autoimmune disease.

Blood tests can be done to determine if a patient has high levels ofantibodies that are indicative of the condition, such as anti-nuclearantibody (ANA) and rheumatoid factor (because SS frequently occurssecondary to rheumatoid arthritis), which are associated with autoimmunediseases. Typical autoimmune disease ANA patterns are SSA/Ro and SSB/La,of which SSB/La is far more specific; SSA/Ro is associated with numerousother autoimmune conditions but are often present in Sjögren's(Franceschini and Cavazzana (2005) “Anti-Ro/SSA and La/SSB antibodies,”Autoimmunity 38 (1): 55-63).

Autoimmune diseases that can be diagnosed by the methods of theinvention include, without limitation, Coeliac disease, diabetesmellitus type 1 (IDDM), lupus erythematosus, systemic lupuserythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome,Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenicpurpura, rheumatoid arthritis (RA), ankylosing spondylitis, Crohnsdisease, dermatomyositis, Goodpasture's syndrome, Guillain-Barrésyndrome (GBS), mixed Connective tissue disease, multiple sclerosis,myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anaemia,psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis,relapsing polychondritis, temporal arteritis, ulcerative colitis,vasculitis and Wegener's granulomatosis

The methods are useful in the diagnosis of a mammal, an animal, or yetfurther a human patient. For the purpose of illustration only, a mammalincludes but is not limited to a human, a simian, a murine, a bovine, anequine, a porcine or an ovine.

In yet another aspect of the above embodiments, the Sjögren's syndromeis an inflammatory autoimmune lacrimal gland disease.

Methods of Treatment

The invention further provides methods of treating subjects havingautoimmune disease or likely to develop autoimmune disease, asidentified above, or ameliorating the symptoms of autoimmune disease inthe subjects. In one embodiment, the method comprises, or alternativelyconsists essentially of or yet further consists of administering aneffective amount of a suitable therapy to the mammal, thereby treatingthe mammal. Non-limiting examples of suitable therapies includecyclosporin, cevimeline, pilocarpine, a nonsteroidal anti-inflammatorydrug, a corticosteroid, an immunosuppressive drug, or adisease-modifying antirheumatic drug. These therapies can be usedseparately or in combination to treat, or alternatively ameliorate thesymptoms of autoimmune disease.

Autoimmune diseases that can be treated by the methods of the inventioninclude, without limitation, Coeliac disease, diabetes mellitus type 1(IDDM), lupus erythematosus, systemic lupus erythematosus (SLE),Sjögren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis,Graves' disease, idiopathic thrombocytopenic purpura, rheumatoidarthritis (RA), ankylosing spondylitis, Crohns disease, dermatomyositis,Goodpasture's syndrome, Guillain-Barré syndrome (GBS), mixed Connectivetissue disease, multiple sclerosis, myasthenia gravis, narcolepsy,pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis,polymyositis, primary biliary cirrhosis, relapsing polychondritis,temporal arteritis, ulcerative colitis, vasculitis and Wegener'sgranulomatosis

Moisture replacement therapies such as artificial tears may ease thesymptoms of dry eyes (some patients with more severe problems usegoggles to increase local humidity or have punctal plugs inserted tohelp retain tears on the ocular surface for a longer time). Cyclosporin(Restasis) is available by prescription to help treat chronic dry eye bysuppressing the inflammation that disrupts tear secretion. Prescriptiondrugs are also available that help to stimulate salivary flow, such ascevimeline and pilocarpine. Nonsteroidal anti-inflammatory drugs can beused to treat musculoskeletal symptoms. Corticosteroids orimmunosuppressive drugs can be prescribed to ameliorate symptoms.Disease-modifying antirheumatic drugs (DMARDs) such as methotrexate canalso be helpful to relieve the patient of the symptoms. Multiplemonoclonal antibodies are currently under investigation (Meijer et al.(2007) Clin Rev Allergy Immunol 32 (3):292-7). For patients with severesymptoms, punctal plugs can be inserted into the lower or upper teardrainage canals of the eyes.

In another aspect, the invention provides a method for treating a mammalsuffering from or at risk of developing autoimmune disease, comprisingadministering to the mammal an effective amount of an agent inhibitingthe expression or activity of a polypeptide selected from the groupCtss, Ctsh, Ctsr, Ctsw or Ctsz.

In one embodiment, the polypeptide is Ctss. In one aspect of theembodiment, the agent is a Ctss antibody. In one aspect of theembodiment, the agent is a small molecule Ctss inhibitor. Small moleculeCtss and Cts1 inhibitors have been designed and used therapeutically.For example, Katunuma et al. disclosed seven inhibitors of the cathepsinL inhibitor Katunuma (CLIK), one of which also inhibited cathepsin S(Katunuma et al. (1999) FEBS Letters 458:6-10).

Other means of inhibiting protein activity or expression can also beused. Non-limiting examples include siRNA, dsRNA, miRNA, antisensepolynucleotide, ribozymes, triplex polynecleotide, antibody and otherinhibitory polypeptides.

siRNA, dsRNA, and miRNA to inhibit protein expression can be designedfollowing procedures known in the art. See, e.g., Dykxhoorn, D. M. andLieberman, J. (2006) Annu. Rev. Biomed. Eng. 8:377-402; Dykxhoorn, D. M.et al. (2006) Gene Therapy 13:541-52; Aagaard, L. and Rossi, J. J.(2007) Adv. Drug Delivery Rev. 59:75-86; de Fougerolles, A. et al.(2007) Nature Reviews Drug Discovery 6:443-53; Krueger, U. et al. (2007)Oligonucleotides 17:237-250; U.S. Patent Application PublicationNo.:2008/0188430; and U.S. Patent Application PublicationNo.:2008/0249055.

Delivery of siRNA, dsRNA or miRNA to a cell can be made with methodsknown in the art. See, e.g., Dykxhoorn, D. M. and Lieberman, J. (2006)Annu Rev. Biomed. Eng. 8:377-402; Dykxhoorn, D. M. et al. (2006) GeneTherapy 13:541-52; Aagaard, L. and Rossi, J. J. (2007) Adv. DrugDelivery Rev. 59:75-86; de Fougerolles, A. et al. (2007) Nature ReviewsDrug Discovery 6:443-53; Krueger, U. et al. (2007) Oligonucleotides17:237-250; U.S. Patent Application Publication No.: 2008/0188430; andU.S. Patent Application Publication No.:2 008/0249055.

Antisense oligonucleotides have nucleotide sequences complementary tothe protein coding or “sense” sequence. Antisense RNA sequences functionas regulators of gene expression by hybridizing to complementary mRNAsequences and arresting translation (Mizuno et al. (1984) PNAS 81:1966;Heywood et al. (1986) Nucleic Acids Res. 14:6771). An antisensepolynucleotide comprising the entire sequence of the target transcriptor any part thereof can be synthesized with methods known in the art.See e.g., Ferretti et al. (1986) PNAS 83:599. The antisensepolynucleotide can be placed into vector constructs, and effectivelyintroduced into cells to inhibit gene expression (Izant et al. (1984)Cell 36:1007). Generally, to assure specific hybridization, theantisense sequence is substantially complementary to the targetsequence. In certain embodiments, the antisense sequence is exactlycomplementary to the target sequence. The antisense polynucleotides mayalso include, however, nucleotide substitutions, additions, deletions,transitions, transpositions, or modifications, or other nucleic acidsequences or non-nucleic acid moieties so long as specific binding tothe relevant target sequence corresponding to the gene is retained as afunctional property of the polynucleotide.

The antisense nucleic acids (DNA, RNA, modified, analogues, and thelike) can be made using any suitable method for producing a nucleicacid, such as the chemical synthesis and recombinant methods disclosedherein and known to one of skill in the art. In one embodiment, forexample, antisense RNA molecules of the invention may be prepared by denovo chemical synthesis or by cloning. For example, an antisense RNA canbe made by inserting (ligating) a gene sequence in reverse orientationoperably linked to a promoter in a vector (e.g., plasmid). Provided thatthe promoter and, preferably termination and polyadenylation signals,are properly positioned, the strand of the inserted sequencecorresponding to the noncoding strand will be transcribed and act as anantisense oligonucleotide of the invention.

It will be appreciated that the oligonucleotides can be made usingnonstandard bases (e.g., other than adenine, cytidine, guanine, thymine,and uridine) or nonstandard backbone structures to provides desirableproperties (e.g., increased nuclease-resistance, tighter-binding,stability or a desired Tm). Techniques for rendering oligonucleotidesnuclease-resistant include those described in PCT Publication WO94/12633. A wide variety of useful modified oligonucleotides may beproduced, including oligonucleotides having a peptide-nucleic acid (PNA)backbone (Nielsen et al. (1991) Science 254:1497) or incorporating2′-O-methyl ribonucleotides, phosphorothioate nucleotides, methylphosphonate nucleotides, phosphotriester nucleotides, phosphorothioatenucleotides, phosphoramidates. Another example of the modification isreplacement of a non-bridging phosphoryl oxygen atom with a sulfur atomwhich increases resistance to nuclease digestion. Increased antisensepolynucleotide stability can also be achieved using molecules with2-methyoxyethyl substituted backbones. See e.g., U.S. Pat. Nos.6,451,991 and 6,900,187.

In another embodiment, ribozymes can be used (see, e.g., Cech (1995)Biotechnology 13:323; and Edgington (1992) Biotechnology 10:256 and Huet al., PCT Publication WO 94/03596). A ribonucleic acid enzyme(“ribozymes”, “RNA enzyme”, or “catalytic RNA”) is an RNA molecule thatcatalyzes a chemical reaction. Many natural ribozymes catalyze eitherthe hydrolysis of one of their own phosphodiester bonds, or thehydrolysis of bonds in other RNAs, but they have also been found tocatalyze the aminotransferase activity of the ribosome. Methods ofmaking and using ribozymes can be found in e.g., U.S. Patent ApplicationPublication No. 2006/0178326.

“Triplex ribozymes” configurations allow for increased target cleavagerelative to conventionally expressed ribozymes. Examples of triplexribozymes include hairpin ribozymes and hammerhead ribozymes. Methods ofmaking and using triplex ribozymes are found in, e.g., Aguino-Jarguin etal. (2008) Oligonucleotides 18(3):213-24 and U.S. Patent ApplicationPublication No. 2005/0260163.

Proteins have been described that have the ability to translocatedesired nucleic acids across a cell membrane. Typically, such proteinshave amphiphilic or hydrophobic subsequences that have the ability toact as membrane-translocating carriers. For example, homeodomainproteins have the ability to translocate across cell membranes. Theshortest internalizable peptide of a homeodomain protein, Antennapedia,was found to be the third helix of the protein, from amino acid position43 to 58 (see, e.g., Prochiantz (1996) Current Opinion in Neurobiology6:629-634. Another subsequence, the h (hydrophobic) domain of signalpeptides, was found to have similar cell membrane translocationcharacteristics (see, e.g., Lin et al. (1995) J. Biol. Chem.270:14255-14258). Such subsequences can be used to translocateoligonucleotides across a cell membrane. Oligonucleotides can beconveniently derivatized with such sequences. For example, a linker canbe used to link the oligonucleotides and the translocation sequence. Anysuitable linker can be used, e.g., a peptide linker or any othersuitable chemical linker.

Antibodies may be raised against any portion of a protein which providesan antigenic epitope. Methods to make and use antibodies to inhibitprotein function are described in e.g., U.S. Pat. No. 7,320,789 and U.S.Patent Application Publication No. 2009/0010929.

In one aspect of the above embodiments, the Sjögren's syndrome is aninflammatory autoimmune lacrimal gland disease. In another aspect, themammal is a human patient.

Kits

As set forth herein, the invention provides diagnostic methods forautoimmune disease. In some embodiments, the methods use probes orantibodies specific for a polypeptide selected from the group Ctss,Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10, Il10ra, Il15, Tnfa, Apo-F,Lcn-2, lactoperoxidase, lactoferrin or lysozyme. Accordingly, theinvention provides kits for performing these methods as well asinstructions for carrying out the methods of this invention such ascollecting tear and/or performing the screen, and/or analyzing theresults, and/or administration of an effective amount of the suitabletherapy.

The test samples used in the diagnostic kits can be tears. Methods forpreparing protein extracts are known in the art and can be readilyadapted in order to obtain a sample which is compatible with the systemutilized. A non-limiting illustrative example is the Schirmer's teststrip discussed above. In one embodiment, the test strip contains or isembedded with a quantitative substrate that is detectably labeled, suchas a fluorometrically labeled antibody, for quantitative detection ofthe one or more polypeptides identified above.

The kits can include all or some of the positive controls, negativecontrols, reagents, probes and antibodies described herein fordetermining the protein expression level or activity level in thesubject.

As amenable, these suggested kit components may be packaged in a mannercustomary for use by those of skill in the art. For example, thesesuggested kit components may be provided in solution or as a liquiddispersion or the like.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Experiment 1

The male NOD mouse is a well-established animal model in which toevaluate the processes of dacryoadenitis and sialoadenitischaracteristic of the human disease. This mouse strain spontaneouslydevelops insulin-dependent diabetes mellitus (IDDM) as well as SjS-likedisease. Dacryoadenitis, which is more severe than sialoadenitis in thismouse model, is fully-manifested by 12-16 weeks. The manifestations andpathological characteristics of the affected LG in the NOD mouseresemble those changes seen in LG of patients suffering fromdacryoadenitis of Sjögren's syndrome. The NOD SCID mouse strain is animmune-incompetent NOD mouse. Prkdc congenic strain can be compared tothe NOD mouse to distinguish events associated with inflammation versusevents characteristic of the strain that are independent of T- andB-cell mediated inflammatory responses. The NOD SCID strain lacksfunctional T, B and NK cells, and is free of exocrine tissuedestruction.

The early pathological events associated with dacryoadenitis in the NODmouse and other disease models include the development of functionalquiescence (e.g., inability of acinar cells to secrete tear proteinsfrom pre-formed secretory vesicles) in LG regions with otherwisenormally-appearing and intact acinar cells, the infiltration ofinflammatory cells from ducts into other regions of the LG to form foci,and the damage of extracellular matrix and other acinar cells by factorsreleased from these infiltrating immune cells. Over time, the healthyacinar cell mass in the LG is replaced by lymphocytic foci and regionsof necrotic and apoptotic cell debris. Some of the early functionalchange in otherwise normally-appearing acini have also been linked toexposure to inflammatory cytokines. For instance, it has been shown thatIL1α and IL1β, constituents of the inflammatory cytokine milieu, canelicit functional changes in release of neurotransmitters frominnervating nerves with the LG responsible for modulation of secretoryresponses, and may also elicit direct functional quiescence when exposedto acinar cells in vitro. However the factors responsible for triggeringthe initial autoimmune inflammatory response that contribute to elevatedcytokine levels in the LG and then progress to elicit this cycle ofdamage are still poorly understood.

The Balb/c mouse can also be used as an experimental model. Because thismodel is induced, one may track disease development, progression andrecovery in the female, which more accurately represents the SjS diseasedemographic in this capacity relative to the NOD model. BALB/c micerecover more quickly from the cytokine injection, within a week, whileC57BL/6 mice display a more gradual recovery of within a few weeks. Onecan start with BALB/c mice. If it becomes necessary to utilize a moreexpanded time period for analysis, C57BL/c mice can be included. Forinjection of female mice, aged 10-12 weeks with IL-1, recombinant humanIL-1α will be used. Both IL-1α and IL-1β elicit comparable effects inthe LG disease model but it is anticipated that it may be possible tosecure some IL-1α from the NCI Preclinical Repository to supplement thestudies. LG in anesthetized mice can be injected with IL-1α (1 μg) in 2μL into each LG. Controls can be injected with comparable volumes ofsaline. Tear fluid collection, tear flow, and corneal, conjunctival andLG integrity and inflammation can be assessed in mice from 1-10 dayspost-injection.

Materials and Methods

Animals and animal procedures: The NOD and BALB/c mouse colonies werebred in the University of Southern California Vivarium using breedingpairs purchased from Taconic (Hudson, N.Y.) and/or Charles RiverLaboratories (Wilmington, Mass.). NOD SCID mice were purchased fromHarlan (Indianapolis, Ind.). Animals were treated and sacrificed inaccordance with policies approved by the University of SouthernCalifornia Institutional Animal Care and Use Committee. The LG wasremoved from mice at different ages, after the animals were euthanizedby intraperitoneal injection with 55 mg of Ketaject and 14 mg ofXylazine per kg of body weight followed by cervical dislocation. Afterbeing removed, LG were either snap frozen and stored in liquid nitrogenfor RNA preparation, or fixed immediately with 4% paraformaldehyde and4% sucrose in PBS for processing and analysis using indirectimmunofluorescence.

Reagents and supplies: The VersaGene RNA Tissue Kit, originally fromGentra Systems, was later purchased from Thermo Fisher Scientific, Inc.(Fair Lawn, N.J.) under the name 5 PRIME PerfectPure RNA Tissue Kit(FP2302410). All materials and reagents for microarray were purchasedfrom Applied Biosystems (ABI, Foster City, Calif.) through theVanderbilt Microarray Shared Resources (VMSR, Vanderbilt University,Nashville, Tenn.). All the following materials and reagents for RT andreal-time PCR were purchased directly from ABI: the high capacity cDNART kit (4368814), TaqMan® universal PCR master mix for real-time PCR(4324018), MicroAmp™ optical 384-well reaction plates (4309849) andMicroAmp™ optical adhesive films (4311971), and TaqMan® gene expressionassays (groups 1 and 2). Group 1 probes include those for the genes ofinterest including Il1b (Mm01336189_mlor Mm00434228_ml), 116 (Mm_ml),1110 (Mm_ml), Il15 (Mm00434210_ml), Tnfa (Mm_ml), Infg (Mm_ml), Ctsh(Mm00514455_ml) Ctss (Mm00457902_ml) and Ctsz (Mm00517697_ml). Group 2probes include those for genes serving as internal controls includingHprt1 (Mm00446968 ml) and Sdha (Mm01352357_ml). Mm followed by 8 digitsrepresents the company assay ID for a TaqMan gene expression assaycorresponding to a specific mRNA locus of a gene.

Rat anti-cathepsin H(CATH) monoclonal antibody (MAB1013) and Goatanti-cathepsin S (CATS) polyclonal antibody (3366-100) for Westernblotting were purchased from R&D Systems, Inc. (Minneapolis, Minn.) andBioVision, Inc. (Mountain View, Calif.) respectively; goat anti-CATHpolyclonal antibody (sc-6497), goat anti-CATS polyclonal antibody(sc-6505), and rat anti-mouse CD14 monoclonal antibody (sc-9150) usedfor immunofluorescent microscopy were purchased from Santa CruzBiotechnology, Inc. (Santa Cruz, Calif.). Rat anti-Lamp2 monoclonalantibody (ab13524) from Abcam USA (Cambridge, Mass.); rat anti-CD68monoclonal antibody (MCA1957GA) from AbD Serotec USA (Raleigh, N.C.);and Rhodamine Red-X-conjugated donkey anti-goat IgG (705-295-147),FITC-conjugated donkey anti-goat IgG (711-095-152) and FITC-conjugateddonkey anti-rat IgG (712-095-150) from Jackson ImmunoResearchLaboratories (West Grove, Pa.) were all used for immunofluorescenceanalysis. Raw264.7 whole cell lysate was used as the positive controlfor CATH and CATS proteins analyzed by Western blotting and waspurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz CA, USA).Carbamylcholine (CCH) was purchased from Sigma (St. Louis, Mo., USA).

Fluorescence-activated cell sorter (FACS) analysis: FACS analysis wasperformed on inflammatory cells that were isolated from LG of 12 weekold BALB/c, NOD and NOD SCID mice (n=5 mice, pooled) according to amodified protocol as described previously (Schenke-Layland et al.,2008). Inflammatory cell subsets were dual-labeled with the followingantibodies: fluorescein isothiocyanate (FITC)-conjugated CD49b/Pan-NK;cyanine-5 (Cy5)-conjugated CD11b (Mac-1) and phycoerythrin(PE)-conjugated Gr1; PE-conjugated B220 and FITC-conjugated CD19; aswell as FITC-conjugated CD4 and PE-conjugated CD8. All these antibodieswere purchased from BD Biosciences/Pharmingen (San Diego, Calif., USA)and used according to the manufacturer's protocol. Nonspecificisotype-matched Cy5-, PE- and FITC-conjugated IgGs served as controls.Staining with 7-amino-actinomycin (7-AAD; BD Pharmingen, San DiegoCalif., USA) was performed to exclude dead cells according to themanufacturer's instructions. Cells were gated properly and a total of10,000 events were acquired for each sample. All analyses were performedusing a BD LSR2 flow cytometer (BD Bioscience, San Jose, Calif., USA).FACS files were exported and analyzed using the FlowJo 8.3.3 software(Tree Star Inc., Ashland, Oreg., USA).

Preparation of total RNA: The preparation was conducted using theVersaGene RNA Tissue Kit or 5 PRIME PerfectPure RNA Tissue Kit at roomtemperature. One to two pairs of LG were taken out from liquid nitrogen,and quickly homogenized on ice using a Brinkman Polytron tissuehomogenizer in lysis buffer. The lysate was filtered through a Pre-Clearspin column by centrifugation. The clarified lysate was passed through apurification column by centrifugation. The RNA-bound membrane wastreated with DNase I. The RNA was eluted into a collection tube from thecolumn with elution buffer. Three LG RNA samples were pooled from 3 micein equal amounts for microarray analysis and real-time RT-PCR. Each RNAsample was prepared for real-time RT-PCR from 3-4 pairs of pooled LGwhen 4-week-old mice were used. All the purified RNA samples were storedat −80° C.

Gene expression microarray analysis: Triplicates of ABI Mouse GenomeSurvey Microarray, AB1700 version 1.0.1 (4382672) were used for eachgroup of mice. Each chip was printed with about 33,000 60-mer oligos asprobes, representing a complete annotated and curated set ofapproximately 32,000 mouse genes from the public and Celera databases.The microarray analysis and the sequential data normalization wereconducted by VMSR. Before microarray, the purity and integrity of RNAswere confirmed by measurement on an Agilent Bioanalyzer according tomanufacturer's manual. In brief, 1 μg of total RNA (about 30 ng mRNA)was used to generate double-stranded cDNA using ABI NanoAmp™ RT-IVTlabeling kit (4365715) according to manufacturer's protocol. The entirecDNA product was used in an IVT reaction to generate digoxigenin(DIG)-labeled cRNA. The cRNA was purified using a kit column andassessed for quality on an Agilent Bioanalyzer. All hybridizationreagents, hybridization controls, wash reagents, and chemiluminescentreagents were provided in the ABI Chemiluminescence Detection Kit(4342142), and the manufacturer's protocol was followed in thesubsequent hybridization procedure. Briefly, the arrays werepre-hybridized with a 1 ml of pre-hybridization mixture for 60 min withagitation at 100 RPM and 55° C. in a hybridization oven. 0.5 ml offragmented DIG-labeled targets mixed with hybridization controls wasadded to the pre-hybridization solution. The arrays were continuallyincubated at 55° C. and agitated at 100 RPM for 16 hr. The arrays werewashed and incubated with anti-DIG-AP antibody for 20 min. Followingantibody washes, the arrays were incubated with ChemiluminescenceEnhancing Solution for 20 min. Substrate for the chemiluminescencereaction was added to each array individually one array at a time. Thearray was immediately imaged on the 1700 Chemiluminescent MicroarrayAnalyzer. The images were assessed for QA/QC and a primary analysis wascompleted by the AB1700 Expression Array System Software (v 1.1.1). Theraw data were normalized using the ABI quantile-based method andfiltered according to the average scores of flags with the analyzer andassociated software.

Reverse transcription (RT) and real-time polymerase chain reaction(PCR): Two reaction steps were carried out with ABI reaction kits andreagents according to the manufacturer's protocols. Briefly, RT reactionwas conducted with 1 μg of RNA per 10 μl of reaction volume at 25° C.for 10 min then 37° C. for 2 hr, and terminated at 85° C. for 5 sec,using the high capacity cDNA RT kit. Real-time PCR was conducted usingan ABI 7900HT Fast Real-Time PCR System. 1 μl of RT product (dilutedwith 3.5 μl of nuclease-free H₂O), 0.5 μl of the TaqMan Assay Mixtureand 5 μl of Universal Master Mix were used in each PCR reaction in atotal volume of 10 μl. Triplicates were run for each assay. The sampleswere preheated at 95° C. for 10 min, followed by 40 cycles of 95° C. for15 sec and 60° C. for 1 min. The PCR reaction with the TaqMan assay forthe house-keeping genes, Hprt1 (hypoxanthinephosphoribosyltransferase 1) or Sdha (succinate dehydrogenase complex,subunit A), were run as internal controls. The recorded data wereanalyzed using the ΔΔCt study calculating function of the ABI softwareSDS 2.1. The fold change (FC) for a specific mRNA was obtained bycalculations as ΔCt=Ct (studied mRNA)−Ct (house keeping gene mRNA), ΔCt(NOD)−ΔCt (BALB/c)=ΔΔCt, and FC(NOD/BALB/c)=2^(ΔΔCt).

Confocal fluorescence microscopy: After removal, LG were incubated inPBS containing 4% paraformaldehyde and 4% sucrose at room temperaturefor 2-3 hr. The gland was transferred to PBS containing 30% sucroseovernight. The glands were embedded into O.C.T. and snap frozen inliquid nitrogen. The blocks were stored at −80° C. prior to tissuesectioning. The blocks were sectioned with a Microm Cryostat(Heidelberg, Germany) into 5 micron thick sections. The slides wereincubated with diluted primary antibody in 1% BSA on the top of thetissue section at 37° C. for 1 hr in a moisturized chamber. Sequentiallydiluted fluorophore-labeled secondary antibodies in 1% BSA andfluorophore-labeled phalloidin (where appropriate) were applied andslides were incubated in the moisturized chamber at 37° C. for 1 hr.Finally, slides were incubated with DAPI in PBS for 5 min, rinsed withwater and mounted with water soluble anti-fade mounting medium(Invitrogen, Carlsbad, Calif.) under a cover slip. During the wholeprocedure, slides were washed with PBS 2-3 times between the treatments.Samples were imaged with a Zeiss LSM 510 Meta NLO confocal/multiphotonimaging system.

Western blotting with LG tissue lysate or tear fluid: Pooled LGs removedfrom 2-3 mice freshly or stored at −80° C. were homogenized with amotor-driven homogenizer in RIPA buffer (150 mM NaCl, 50 mM Tris-C1,0.5% sodium deoxycholate, 0.5 mM EDTA, 0.1% TX-100, 1% NP-40) containingprotease inhibitors in a tissue: buffer ratio of 1:5 (w/v). Theresulting homogenate was clarified by centrifugation at 10,000 rpm at 4°C. for 10 min. The supernatant was collected and stored at −80° C. Analiquot of the supernatant was mixed with SDS gel loading buffer andheated at 92° C. for 5 min for the subsequent analysis.

For tear collection, the mouse was anesthetized as described above. Themouse LG was exposed by a small incision along an axis defined by theouter junction of the eyelid and the ear, then covered with a layer offine cellulose mesh (Kimwipe®) cut into a similar size as the gland. TheLG was stimulated by adding the agonist carbamylcholine (CCH) (5 μL, 10μM) onto the mesh on the top of the gland, and tear fluid was collectedwith glass capillaries at the medial canthus of the eye with care takennot to touch the cornea. Each eye was stimulated two times, in a totalcollection time of 10 min per eye. The collected tear fluid wastransferred from the capillaries to an Eppendorf tube containingprotease inhibitors, measured for precise volume, mixed with SDS gelloading buffer, pooled when necessary, and heated at 92° C. for 5 min.

Tissue lysate containing 100 μg of total proteins or 1 μl of tear fluidwere loaded to each well and resolved on 10-12% SDS PAGE. The membraneswere scanned using a LI-COR Odyssey Infrared Imaging System.

Measurement of enzymatic activity of cathepsins: For activitymeasurements in LG lysate, freshly collected LG pairs from eachindividual mouse, either post-stimulation after topical CCH for tearcollection or without stimulation, were homogenized with BrinkmanPolytron tissue homogenizer on ice in CS Cell Lysis Buffer (1 mgtissue/5 μl buffer) provided in the Cathepsin S Activity Assay Kit(Biovision, Inc. Mountain View, Calif.). The same number of NOD andBALB/c mice were used in each experiment. The homogenate was clarifiedby centrifugation at 10,000×g, 4° C. for 10 min. The resulting lysatewas either used immediately or stored at −80° C. for later use.

For activity measurements in tears, mice were anesthetized as describedand tear fluid was collected from paired 12-week-old male NOD and BALB/cmice, matched into pairs according to age and sex. The mice were placedresting on their sides under a Motic SMZ-140 dissection microscope(Xiamen, China). The LG was exposed by a small incision along an axisdefined bye the outer junction of the eyelid and the ear and connectivetissue capsule enclosing the gland was carefully opened and removed fromthe upper surface of the gland to which a layer of fine cellulose mesh(Kimwipe®) cut into the shape of the gland but slightly smaller wasapplied. The ocular surface was washed with AK-Rinse Eye IrrigatingSolution (Akorn, Abita Spring, La.). The LG was stimulated by adding theagonist CCH (3 μL, 50 μM) topically to the gland and tear fluid wascollected by carefully applying a 2 μL, microcaps pipette, (Drummond,Broomall, Pa.) at the medial canthus of the eye, for 5 min. Care wastaken not to touch the cornea. Each eye was stimulated with CCH threetimes, resulting in a total collection time of 15 min per eye. Themicrocaps were emptied into sterile vials by the aspirator supplied bythe manufacturer. The tears collected from both eyes of the same mousewere pooled and immediately analyzed for CATS activity.

CATS activity in LG lysate and tear fluid samples were analyzed usingthe Cathepsin S Activity Assay Kit. The collected tear fluid of wholevolume from each mouse or 10 μg of LG lysate was diluted to constitutethe reaction mixture of 100 μL, with or without inhibitor according tothe manufacturer's instructions. The reaction was incubated at 37° C.for 1, 2, and 18 hr. The concentration of resulting fluorescent productswas measured using a fluorimeter with 505 nm emission filter. CATHactivity was assayed with the Cathepsin H Activity Assay Kit (BioVision,Inc.). The procedure was similar to that for CATS activity assay exceptthat only LG homogenate and not tears were analyzed in this assay.

Results

Gene expression profiles of cathepsin family members and otherinflammatory factors in LGs of NOD and BALB/c mice. Severe extracellularmatrix degradation and immune cell infiltration are prominent featuresof LGs in male NOD mouse LG aged 12-18 weeks. Cathepsins are a majorcategory of proteases responsible for regulation of extracellularmatrix; in fact one of their roles as tear secretory proteins is toregulate extracellular matrix homeostasis. Gene expression profiles wereanalyzed to determine possible alterations in their expression in maleNOD mouse LG, using gene expression microarray analysis to investigateif cathepsin family members contributed to these pathologic eventsassociated with immune cell infiltration. The results are presented inTable 1. The hybridization signals for mRNAs of cathepsins H (Ctsh), R(Ctsr), S (Ctss), W (Ctsw), and Z (Ctsz) were elevated in the LG of NODmice compared to the BALB/c controls. There was no difference for therest of the cathepsin family members except cathepsin K (Ctsk) whichshowed expression that was 40% as high as the BALB/c control.

Macrophage-expressed cytokines and their receptors were also examinedfor their mRNA levels in the LGs of NOD mice relative to the BALB/ccontrols. However, only a subset of these cytokines and receptors weredetected by microarray due to the relative insensitivity of thistechnique to low abundance mRNAs as shown in Table 2. The mRNA levels ofinterferon-γ, interleukin-10 receptor α and tumor necrosis factor α wereclearly also higher in the LGs of male NOD mice than that of matchedBALB/c mice.

Data validation and expanded investigation of gene expression. Theresults of microarray were validated by real-time RT-PCR which evaluatedthe expression levels of cathepsin family members and cytokines ofinterest. Beside the total RNAs from the LGs of 12-week-old male NOD andBALB/c mice, total RNAs from age matched NOD SCID mice.

TABLE 1 Differentially expressed cathepsin family members in LGs of NODmice versus BALB/c mice characterized by microarray analysis Change GeneNCBI Accession FC (NOD/BALB) P Value in NOD Ctsb BC006656 1.0 0.3330 nochange Ctsc NM_009982 1.2 0.0624 no change Ctsd NM_009983 1.0 0.3311 nochange Ctsf NM_019861 0.7 0.0006 no change Ctsh NM_007801 2.1 0.0015increase Ctsk NM_007802 0.4 0.1128 may decrease Ctsl NM_009984 0.90.4897 no change Ctso NM_177662 1.4 0.1111 no change Ctsr NM_020284 6.90.0015 increase Ctss NM_021281 4.4 1.7E−07 increase Ctsw NM_009985 3.10.0230 increase Ctsz NM_022325 1.8 0.0029 may increase FC represents thefold change obtained by comparing the hybridization signal of NOD mouseLG to the signal from BALB/c mouse LG (NOD/BALB/c) after normalization.The full names for the gene symbols listed in this table are:Cts(letter), genes for cathepsin family members.

TABLE 2 Increased mRNA levels of cytokines and proinflammatory factorsin LG from NOD versus BALB/c mice characterized by microarray analysisChange Gene NCBI Accession FC (NOD/BALB) P Value in NOD Ifng NM_00833714.5  1.2E−06 increase Il1b NM_008361.3 — — under detection Il6NM_031168.1 — — under detection Il6ra NM_010559 2.6 0.1740 may increaseIl10 NM_010548 2.6 0.0636 may increase Il10ra NM_008348 6.1 0.0014increase Il15 NM_008357 1.8 0.3883 may increase Tnfa NM_013693 4.60.0003 increase FC represents the fold change obtained by comparing thehybridization signal of NOD mice to the signal of BALB/c mice(NOD/BALB/c) after normalization.

TABLE 3 FACS analysis of inflammatory cells isolated from LGs of BALB/c,NOD and NOD SCID male mice. Lineage Marker BALB/c NOD NOD SCID B220+CD19− 6.8% 3.9% 5.1% B220+ CD19+ 4.1% 34.0% 0.8% CD4+ CD8− 3.1% 13.0%3.1% CD4− CD8+ 2.1% 7.9% 2.0% CD11b+ GR1− 7.0% 25.0% 11.0% CD11b+ GR1+2.0% 3.5% 1.7% CD11b− GR1+ 4.2% 6.9% 3.0% Pan-NK 3.6% 5.9% 2.2%

5 male mice of each strain each aged 12 weeks were used for LGcollection and isolation of interstitial inflammatory cells aspreviously described. B220+ CD19−, characterized as early B lymphoidprogenitor cell; B220+ CD19+, B lymphoid progenitor cell; CD4+ CD8−,mature T helper cell; CD4−CD8+, mature cytotoxic T cell; CD11b+GR1—,macrophage; CD11b+GR1+, myeloid immunoregulatory cell; CD11b− GR1+,granulocytes; Pan-NK, NK cell. Percentage of each cell lineage wasobtained by the cell number of a specific cell subset divided by totalcell counts analyzed, then multiplied by 100%.

Female NOD and BALB/c mice, and 4-week-old NOD and BALB/c mice of bothgenders were also analyzed concomitantly. The results are summarized inFIG. 1. The expressions of the cytokines which were not detected bymicroarray, possibly due to low abundance, were also re-evaluated bythis method. Consistent with the microarray analysis, the mRNA levels ofCtsh, Ctss and Ctsz in the 12 week old male NOD mouse LG were markedlyhigher than in the BALB/c mice as shown in FIG. 1A; all themacrophage-produced cytokines tested also showed markedly increasedexpression in the LG of NOD mice to different extents as shown in FIG.1C. Interestingly, the mRNAs levels of the obesity-induced proteinsCtsh, Ctsz, Ctss, 11-6 and INF-α were all higher in NOD SCID mice thanin BALB/c mice although the levels in NOD SCID mice were still lowerthan those detected in NOD mice (FIG. 1A); NOD SCID mice, like NOD mice,exhibit notable lipid deposition in the LG of the male mice. 11-10 wasthe only gene which exhibited equivalent elevated expression betweenmale NOD and NOD SCID mouse LG (FIG. 1C). Comparison of gene expressionlevels in mice aged 4 weeks showed little to no changes in these samemarkers (FIGS. 1B and 1D). This age of 4 weeks is prior to the onset oflipid deposition and accumulation.

Macrophages are prominent infiltrating cells in diseased LG. A previousstudy has revealed the presence of various types of infiltrating immunecells including macrophages, neutrophilic and eosinophilic granulocytes,B-cells and T-cells within the LG of NOD mice at 18 weeks of age. Theexpression profiles of cathepsins and cytokines by real-time RT-PCRmeasured here also suggested the activation of macrophages in NOD mouseLG. To understand the role macrophages play in this inflammatoryautoimmune disorder in this disease model, lineage classification wasperformed by FACS analysis with prepared immune cell populations frompooled LGs from 12-week-old male NOD, NOD SCID and BALB/c mice.Proportions of B-cells (B220+ CD19+), T-cells (CD4+ and CD8+),macrophages (CD11b+) and other immune cells out of the whole immune cellpopulation counted in the LGs of three strains are listed in Table 3.The result showed that macrophages constitute a major population (25%),the second large population after the B cells in NOD mouse LG. NOD SCIDmice lack T and B cells and have only partial competence in the functionof myeloid cells. Consistent with this, there were significantly lownumbers of B-cells (B220+CD19+) and T-cells (CD4+ and CD8+) detectedfrom NOD SCID mouse LG compared to that from NOD mice. The numbers ofthese cell types were even lower than that from BALB/c mouse LG; on theother hand, the macrophage population within the LG of NOD SCID mice wasincreased to 11% in contrast to 7% in the LG of BALB/c mice. This numberwas still lower than that in matched NOD mouse LG due to the lack ofstimulation by and communication with lymphocytes.

Characterization of CATS protein distribution in LG within macrophagesand acinar cells. Previous studies demonstrated that CATS participatesin both antigen presentation and normal cellular protein turnover andalso degrades extracellular matrix in cancers. The extensiveextracellular matrix degradation and immune cell infiltration combinedwith the gene expression profiling result from the current studyindicated that the increased expression of CATS may directly contributeto LG destruction by degrading the extracellular matrix or alternativelythat it may indirectly contribute to LG destruction by stimulatingneoautoantigen presentation and consequent lymphocyte proliferation.Hence, the localization and abundance of CATS protein in LG wasinvestigated using immunofluorescence microscopy. The results showedthat CATS was present in populations of CD68-positive and -negativeinfiltrating cells (FIG. 2) within the LG. It was also noted that thedistribution pattern of CATS was very similar to that of CATH. BothCATS-positive cells were within the connective tissues surrounding theLG of the all three strains, whereas CATS-enriched cells in the interiorregion of the gland were only detected in LG from NOD and NOD SCID mice.Additionally, CATS-positive cells were seen among the lymphocytic fociin the LG of NOD mice.

CATS immunofluorescence was also detected within the acinar cells inaddition to the macrophage and the other cell types described above. Thenumber and size of Lamp2-positive late endosomes/lysosome and theabundance of the CATS protein appeared to be markedly increased inacinar cells from NOD (FIG. 3A) and NOD SCID (FIG. 3B) mice relative tothe amounts in acinar cells from LG from matched BALB/c mice (FIG. 3C).CATS was also detected in the organelles in the subapical regionsurrounding the lumen of acinar cells from NOD (FIG. 3D) and NOD SCID(FIG. 3E) mice in contrast to the solely basolateral punctate labelingfor CATS in the acinar cells from BALB/c mice (FIG. 3F).

Increased abundance and activity of CATS in NOD mouse LG lysates andtears. The increased copy number of Ctss mRNA, the detection ofadditional CATS-positive cells in NOD mouse LG, and the detection ofincreased CATS immunofluorescence within subapical compartments inacinar cells described above all suggest an increased protein abundanceand catalytic activity of CATS in LG under these pathologicalconditions. The CATS abundance was thus compared in LG of NOD mice tothat of BALB/c mice by Western blotting analysis (FIG. 4A).Consistently, a clear 24 kD MW protein band corresponding to themolecular weight of active form of CATS in the gland lysate from NODmice was detected (4A) but a much weaker to no band at the same positionin LG lysate from BALB/c mice (4A). Enzymatic activity assays were alsoconducted for comparison of the CATS activities in LG between the twostrains (FIG. 4B). The result showed the average CATS activity from NODmouse LG lysates was significantly greater than that from the controlBALB/c mice LG lysates.

The evident redistribution of CATS immunofluorescence into apparentsubapical secretory vesicle-like organelles as well as within the lumenain the acinar cells suggested that CATS may be actively secreted at theapical membrane into the tear fluid in NOD mice. Hence the enzymaticactivities of the tears were measured in the absence or presence ofspecific inhibitor, in parallel with the LG lysate. The resultsdemonstrated significantly higher CATS activities in tear fluid of NODmice relative to those of BALB/c mice upon stimulation of the LG withthe agonist, CCH (FIG. 5). Consistent with the testing results from LGlysates, the average enzymatic activity of the tears collected wasmeasured from the stimulated glands of NOD mice versus from that ofBALB/c mice.

Cathepsin D, like CATH and CATS, is another member of peptidase C1family expressed in LG (Table 1). Using this activity as a controlactivity with expression unchanged in the NOD strain relative to theBALB/c strain, no differences in either catalytic activities or glandlysates or tears was detected between the two mouse strains, consistentwith the result of the microarray (FIG. 5C).

Characterization of CATH protein within macrophages but not acinar cellsin LG. CATH is defined as an aminopeptidase (notably, cleaving Arg-|-Xaabonds) as well as an endopeptidase. Its cellular function is somewhatobscure to date. Its mRNA was markedly elevated in the NOD mouse LG.Immunofluorescent microscopy was performed to localize the cellsproducing CATH protein. The results are shown in FIG. 6. Similar to thedistribution of CATS, the CATH protein was observed in some cells ininterstitial and elastic tissue within the sac surrounding the LG inNOD, NOD SCID and BALB/c mice (FIGS. 6A, D and G). The CATH-positivecells were also enriched at the intercellular space between the acinifrom NOD and NOD SCID mice but not from BALB/c mice (FIGS. 6F and I). Inaddition, CATH-positive cells were observed among the infiltrating fociin the LG of NOD mice (I). Some but not all CATH-positive cells werealso positive for CD68 and these cells were seen either at theextracellular space or within the foci (FIG. 6C, K, L). Unlike CATS, noCATH protein was detected in the acinar cell.

Western blotting analysis was conducted to investigate if the abundanceof CATH protein also increased in parallel with the elevated mRNA levelsin NOD mouse LG. The result showed that there were two protein bandsrecognized by the anti-CATH antibody with approximate molecular weightsof 37 and 27 kD, corresponding to the full length protein and the activecleaved form of CATH, respectively (FIG. 7A). The abundance of CATH,especially the active form, was significantly higher in the lysates ofLGs from NOD mice than from BALB/c mice.

The enzymatic activity of CATH was also determined as shown in FIG. 7B.Significantly elevated catalytic activity of the enzyme was detected inLG lysates from 12-week-old male NOD mice versus that from the matchedBALB/c mice. Applicants noted that CATH remained catalytically activefor an extended period of time, especially in the assay with NOD mouseLG lysates, in comparison to CATS and CATD. Additionally, the catalyzingactivity was only partially inhibited by the CATH inhibitor (rightpanel), indicating a likely contribution of the partial activity byother non-CATH enzyme(s) or limited inhibitory ability of the inhibitor.

DISCUSSION

The experiment described above reports, for the first time, thesignificant upregulation of CATS expression and activity in the LGduring development of autoimmune inflammatory disease. Applicants' datafurther suggests, as specified below, that CATS expression isupregulated in both the infiltrating immune cells as well as in theacinar cells that constitute the bulk of the gland, suggesting a complexrole for CATS in etiology of disease.

Consistent with gene expression analysis, CATS-positive cells aredetected more abundantly in the NOD mouse LG than in the BALB/c mouseLG. CATS protein was detected in a vast number of the infiltrating cellsin the NOD mouse LG within foci as well as at the LG periphery, but wasonly detected in some macrophage-like cells in the peripheral connectivetissue-enriched areas of the BALB/c mouse LG. While many of theCATS-enriched cells within inflammatory cell foci in the NOD mouse LGwere CATS+/CD68+, confirming their identity as macrophages, aconsiderable number of CATS-positive cells were CD68-negative,suggesting the presence of other antigen presenting cells (APC).Additionally, a notable proportion of CATS positive cells were extremelylysosome rich as evidenced by their enrichment in Lamp2 staining. Theseobservations are consistent with previously established mechanisms forthe role of CATS in antigen presentation and cellular protein maturationin macrophages and other APCs, which contribute to activation of T-, B-and other lymphocytes.

Beside the CATS-positive immune cells, CATS immunofluorescence and, byextension, protein abundance was also significantly increased in theacinar cells from NOD and NOD SCID mouse LG versus that from BALB/cmice. The acinar cells constitute approximately 85% of the cell mass ofthe LG and are largely responsible for production and release ofsecretory proteins into the tears. They therefore maintain an abundantarray of mature secretory vesicles within their subapical cytoplasm. Asubset of CATS-enriched organelles within the acinar cells wassignificantly co-localized with markers within the acini. These findingssuggested stimulated biogenesis of lysosomal-like organelles in acinifrom NOD mouse LG. A subset of CATS was also detected within very largesecretory vesicle-like organelles localized in the sub-apical region, aswell as within the lumena of the acini of NOD and NOD SCID mice, but notin the acini of BALB/c mice, indicating altered protein sorting andapical secretion for CATS in the NOD background. This finding wasverified, in the NOD mouse, by the detection of increased CATS activityin tears relative to the BALB/c control strain. Routinely, newlysynthesized lysosomal proteins such as CATS are processed in theendoplasmic reticulum (ER) and Golgi apparatus to the trans-Golginetwork (TGN), then are actively sorted to lysosomes via late endosomesas the terminal destination. This sorting path appears to be partiallyaltered in NOD mouse with a diversion of some of the upregulated CATSinto the regulated apical secretory pathway. This missorting may arisefrom one of two possibilities: 1) CATS overproduction may saturate thenormal sorting pathways in the TGN such as mannose-6-phosphate receptorsand others that sequester lysosomal proteins into cargo vesiclesdestined for lysosomes, and thus the extra CATS may traffic through adefault pathway into mature secretory vesicles; 2) CATS may be activelymissorted into this path due to a fundamental abnormality in thecellular trafficking system of NOD (and NOD-SCID) mice.

While CATD is known to be an established lysosomal resident protein, itis also known to be a component of normal tear fluid, consistent withApplicants' unpublished proteomic data in the mouse. Gene expressionmicroarray showed no change in CATD expression at the mRNA level, andthe corresponding enzymatic analyses showed no changes in either LGtissue or tears from NOD mice compared to the control, despite thesignificant elevation of CATS activity. Therefore it appears that CATDis not involved in nor influenced by the disease progression in NODmouse LG. This result is supportive of the theory that the intracellularsorting of lysosomal proteins is regulated by the abundance of eachrespective protein, since only the upregulated CATS and not thenormally-expressed CATD are missorted to secretory vesicles. However,membrane trafficking is highly dependent on lipid membrane composition,and lipid-enriched subdomains or “lipid rafts” are known to serve assignaling platforms and to mediate specific internalization events suchas caveolar endocytosis, so an alternative theory is consistent with thelipid metabolic abnormalities in the acinar cells from NOD mice,particularly if different sorting processes are involved in capture ofCATD and CATS to lysosomes. Regardless of cause, this collectiveobservations of increased lysosomal abundance in parallel withmissorting of lysosomal proteins into the regulated secretory pathwayeffect demonstrates global changes in protein sorting and processingwithin the acinar cells that may contribute significantly to pathology.The increased CATS and lysosomal activity suggests enhanced and possiblyabnormal protein degradation and processing which may enhance CATSproduction of neoautoantigens in the altered lysosomal pathway of theacinar cells, while enhanced CATS activity in tears may result in tearprotein degradation and also extracellular matrix damage to the ocularsurface.

In addition to CATS, the spectrum of upregulated inflammatory cytokinesthat accompany CATS upregulation in obesity were also detected in theNOD mouse LG. Without being bound by theory, Applicants hypothesize thatthese cytokines are largely produced by infiltrating macrophages, basedon the following reasoning. The LG of NOD SCID mice still retainsmacrophages at an increased number relative to BALB/c mice although notas many as in the NOD mice, while it lacks T and B lymphocytes. Inparallel, increased mRNAs of Tnfa, Il6 and Il10 were detected in NODSCID mouse LG relative to BALB/c mice although to a less extent comparedwith NOD mice. These results suggest that the macrophages present in theLG of NOD mice, compared to NOD SCID mice, are further stimulated bylymphocytes resulting in the increase in their number in parallel withthe more vigorous inflammatory response.

CATH protein, also significantly upregulated in NOD mouse LG relative toBALB/C mouse LG, was exclusively detected in non-acinar cells includingcells migrating into the extracellular space between acini as wellwithin infiltrating foci. Applicants also noted a corresponding increasein CATH enzymatic activity within NOD LG lysates. Persistent conversionof the substrates into products in the proteolytic activity assay duringan 18 hr extended time course indicates that CATH has a stable and longlasting activity in vivo compared to CATS. Partial co-localization ofCATH immunofluoresence with that of CD68 indicates some of CATH-positivecells are macrophages. The physical locations of CATH suggest itsinvolvement in macrophages and other inflammatory cell functions.Previous studies reported that this enzyme is secreted from neutrophilsand participates in the degradation of extracellular matrix. Theextensive loss of extracellular matrix within the NOD mouse LG that haspreviously been reported thus renders CATH a candidate for thispathological event in the NOD mouse model.

The increased protease activity of CATS and CATH within the LG hasmultiple possible consequences. First, the increase in CATS-enrichedlysosomes labeled with Lamp2 suggests that lysosomal degradativecapacity may be enhanced, thus raising the possibility that proteasedegradation of proteins in lysosomes becames abnormal. Previous work hassuggested that alterations in proteolytic activity may expose crypticepitopes on otherwise tolerated self-proteins, which may be effluxed orrecycled into the interstitium from the late endosomal compartmentswhere they may encounter increased CATS. Cryptic epitopes in theinterstitium may encounter primed APC and macrophages, thus potentiatingautoimmunity. Likewise, CATS activity on the ocular surface and CATHactivity within the tissue may promote loss of extracellular matrix inthe ocular surface system.

The detection of proteolytic activity of CATS in the tear fluid of NODmouse in parallel with the detection of CATS immunoreactivity insubapical secretory vesicles shows that this enzyme is secreted fromacinar cells. This fact suggests the possibility that the CATS in tearsmay digest certain protein components of the cornea, therefore damagethe integrity of the ocular surface as well as enhancing sensory inputfrom the cornea to the LG, an event that has been previously linked tothe functional quiescence that characterizes the LG in SjS. Sincecathepsins have collagenase/elastinase activity, the presence of bothCATS and CATH in macrophages and other interstitial cells may alsodegrade tissue extracellular matrix, thus expediting the infiltration ofimmune cells and the loss of secreting function. Targeting CATS and CATHmay therefore constitute alternative therapeutic strategies in thetreatment of chronic autoimmune dacryoadenitis associated with SjS.

In summary, the NOD mouse model is an established model of SjS-likechronic autoimmune dacryoadenitis. Specific cathepsin family members andcytokines are upregulated during development and progression of diseasein this mouse model, with a profile comparable to those changes seen inobesity, suggesting that the lipid deposition plays a causal role in theautoimmune inflammatory response. The profile of increased cathepsinprotease expression and distribution within macrophages, other APCs andeven within acinar cells suggest a complex role for these proteases ininitiation and progression of autoimmunity. Overexpressed CATS secretedinto the tear fluid from NOD mice reflects the initiation of disease andthe consequent changes in LG function and thus may serve as a biomarkerfor diagnosis of autoimmune dacryoadenitis in human. CATS and CATH mayalso be considered as potential targets for alternative therapeuticapproaches to treat and prevent progression of SjS, particularly if wayscan be identified to specifically target such inhibitors to the sites ofinterest within the LG.

Experiment 2

SjS biomarkers in tears are correlated with the severity of inflammatoryautoimmune lacrimal gland disease. Applicants can utilize three uniquetear biomarkers for SjS, CtsS, Apo-F and Lcn-2, that can be expressed intears at levels proportional to the extent of inflammatory autoimmune LGdisease manifested in the source of the tears. In mouse and human tears,levels of these three biomarkers are quantified, and submitted to beincreased in disease, in parallel with three tear secretory proteins,lactoferrin, lactoperoxidase and lysozyme, submitted to be generallydecreased in SjS as well as other KCS disorders.

The NOD mouse and the IL1-injected BALB/c mouse represent two models ofinflammatory autoimmune LG disease. Tears and ocular tissue samples arecollected from these models at intervals relevant to disease developmentand progression. In parallel, the extent of inflammatory autoimmune LGdisease is quantified by analysis of cornea, conjunctiva and LG. Allbiomarkers can additionally be screened in tears from populations ofcontrol and primary SjS patients with established clinical symptoms.Analysis of biomarkers in tears can use standard Western blotting, ELISAor biochemical tests.

SjS patients. Patients with diagnosed primary SjS, according to therevised version of the European criteria proposed by theAmerican-European Consensus Group, will be recruited from the USC CorneaClinic, Rheumatology Clinic or LAC+USC with the aid of collaboratingphysicians. Since the majority of our SjS patients do not producesufficient tears, these may require an “eye-wash”. Before tearcollection, SjS patients (and controls) will undergo a thorough eyeexam. This information can be used for correlation studies. Briefly, adrop of local anesthetic (to collect unstimulated or basal tears) isfirst applied to the ocular surface followed by 2 drops of saline(because most SjS patients have no visible tears). To be consistent,tears should be collected from control subjects following the sameprotocol. The subjects are subjected to a Schirmer's test and teststrips will be saved for collection of disease biomarkers. Tears areeluted into assay buffers for biochemical measurement of enzyme activityand/or analysis of protein content by bioassay or Western blotting. Thismethod of collection has yielded tears from a variety of patients can beeluted for biochemical studies including resolution of proteins onSDS-PAGE gels (FIG. 10) and that exhibit a wide variation in Cathepsin Sactivity that appears to be related to disease state.

Potential Sjs Biomarkers to be Analyzed in Tear Fluid—Predicted IncreaseAssociated With Disease

CtsS is a type of cathepsin, a major class of lysosomal proteases. Theyare established effectors of protein degradation within the lysosomes,and their activity is critical to additional activities includingprecursor protein activation, MHC II-mediated antigen presentation,reproduction and apoptosis (Turk, et al. (2001) EMBO J. 20(17):4629-33).Several cathepsins are expressed in LG, but CtsS in particular has acompelling history related to autoimmunity. CtsS is highly expressed inantigen-presenting cells and is implicated in MHC II-mediated antigenpresentation. It is implicated in the pathogenesis of degenerativeimmune diseases including chronic inflammation and rheumatoid arthritis.CtsS is also upregulated in response to lipid deposition, a feature ofLG pathology strongly associated with inflammatory autoimmune disease inthe NOD mouse LG. CtsS activity has been targeted therapeutically in amouse model of SjS; SG and LG lymphocytic infiltration was prevented byprior administration of a CtsS inhibitor to SG. Significantly increasedactivity of CtsS in LG sections and tears from diseased male NOD mice isdemonstrated. CtsS also shows an 11-fold increase in gene expression inmale NOD mouse LG, relative to age-matched BALB/c mouse LG. Thebiochemical spectrophometric assay illustrated in FIG. 8B can beutilized for measurements of CtsS activity in tears.

Apo-F is a constituent of HDL and LDL, and can be associated withcholesterol and cholesterol esters in lipoproteins. Very little is knownabout its biological function. Recent findings in the LG suggest that itmay normally modulate cholesterol influx and/or excretion in LG at thebasolateral membrane, and that its extensive upregulation, possibly inresponse to metabolic abnormalities and/or inflammation in diseasedLGAC, may lead to its mis-sorting and apical excretion into tears. Apo-Fshows an 53-fold increase in gene expression in male NOD mouse LG,relative to age-matched BALB/c mouse LG, and its content in male NODmouse tears is markedly increased relative to virtually undetectablelevels in tears from age-matched BALB/c controls. Its presence andabundance in tears will be quantified by Western blotting in theseproposed studies. To supplement mouse-specific Apo-F antibody, which canbe used to probe mouse tears in healthy and diseased mice, ahuman-specific Apo-F antibody can be utilized. Applicants have obtainedsuch from Drs. Bill Lagor and Daniel Rador (University of Pennsylvania)that is appropriate for immunoblotting of human tears from healthy andprimary SjS patients.

Lcn-2, also called neutrophil gelatinase-associated lipocalin, isexpressed in both mice and humans, and has been shown to be highlyexpressed in pancreatic islets, bone marrow, and SG. Until now itsexpression in LG has not been investigated but microarray data suggestmoderate to high levels of expression. It is implicated in diversebioprocesses including iron-siderophore binding in bacterial infectionsas a component of the innate immune system, modulation of inflammation,and is a marker closely related to obesity and insulin resistance (Flo,et al. (2004) Nature 432(7019):917-21, 13). FIG. 9 shows the apparentincrease in the immunofluorescence associated with Lcn-2 in diseasedmale NOD mouse LG and its redistribution to mature secretory vesiclesand within the lumena, suggesting drainage into tears. Theseobservations, in parallel with the 2.4× upregulation of Lcn-2 indiseased NOD mouse LG relative to BALB/c mouse LG, suggests its utilityas a putative tear biomarker. Like CtsS, its upregulation in diseasedNOD mouse LG may be related to tissue lipid deposition whichcharacterizes this animal model as well as SjS patients. Lcn-2 has alsobeen reported to be upregulated under conditions of inflammation and byIL-1β and IFNγ cytokines. Applicants have had difficulty reliablydetecting a signal for Lcn-2 by Western blotting in tears of NOD mice,likely due to relative antibody insensitivity. This is one of thereasons that this biomarker was chosen for development of an ELISAcapture assay in as discussed above, as well as the more sensitivecyclotide-based assay also discussed above.

Potential Sjs Biomarkers to be Analyzed in Tear Fluid-Predicted DecreaseAssociated With Disease

Lactoperoxidase, Lactoperoxidase is a secreted glycoprotein member ofthe peroxidase family. It has antimicrobial properties due to itsability to sequester calcium, iron and heme B, and also supplies apotent antioxidant activity to the tears through its ability to formreactive bromine and iodine species through hydrogen peroxide oxidationof halides (Ghibaudi & E. Laurenti (2003) Eur J. Biochem.270(22):4403-12). Its activity can be measured through simplespectrophotometric assays.

Lactoferrin, is also a secretory glycoprotein that is a member of thetransferrin family. It is the principal iron-binding protein in milk andbodily secretions, providing a potent antimicrobial activity as part ofthe non-specific immune system. It has a broad range of activityincluding regulation of iron homeostasis, host defense, regulation ofgrowth and differentiation and protection against cell transformation(Weinberg (2007) Curr Pharm Des. 13(8):801-11; Zimecki et al. (2007) J.Exp Ther. Oncol. 6(2):89-106). It can be assayed by ELISA usingcommercially available antibodies, or immuno-immobilization followed byenzymatic activity assays using commercially available kits.

Lysozyme, is a secretory protein that is ubiquitously present in humanserum, urine, tears and milk (Zimecki et al. (2007) J. Exp Ther. Oncol.6(2):89-106). It hydrolyzes glycosyl linkages present in themucopolysaccharide cell wall of diverse organisms. It can be assayedusing a simple fluorescence-based enzymatic activity assay that iscommercially available.

Collection and Analysis of Tissues and Fluids:

In mouse models, a comprehensive series of established assays can beconducted to quantify the extent of disease development and progressionincluding an analysis of tear flow and corneal integrity in the liveanimal, as well as the isolation and analysis of tissue integrity andinflammation in LG, cornea and conjunctiva after tear collection andsacrifice of the animal. The relationship between the basic assays thatwill be conducted and the designation of autoimmune inflammatory diseasedevelopment in mouse models as mild, moderate or severe is shown inTable 1.

Tear flow and corneal integrity in live mice: For measurement of tearproduction and corneal integrity in live mice, one means is cottonthreads pre-impregnated with phenol red that turn red upon contact withtear fluid. Mice can be anesthetized with 2-3% isoflurane. The thread isinserted into the lateral canthus for 30 sec with care not to touch thecornea. Wetting of the thread is evaluated under the magnifier by atleast 2 evaluators and averaged in millimeters. After the measurementwith the cotton thread or in separate populations of anesthetized mice,corneas are evaluated for any pathological signs as reflected byfluorescein staining in a dark room. One μL of freshly made 1% sodiumfluorescein is carefully applied to the lateral canthus and the eyelidsmanually blinked 3× to spread the solution. Excess solution is removedby gently applying a Kimwipe tissue to the lateral canthus and cornealfluorescein staining is immediately (within 30 sec of addition)evaluated and photographed with a Motic microscope equipped with a CCDcamera using a cobalt blue light. Images are evaluated and graded by astandard protocol. These tests can be conducted on the same mice thatwill subsequently be used for collection of tear fluid, LG and ocularsurface tissue below, as long as the mice are allowed to recover fromanesthesia for several hrs following the isoflurane.

Analysis of biomarker content of tear fluid: For collection of lacrimalfluid, mice are anesthetized with ketamine/xylazine, placed on a heatedsurgical table and immobilized with non-penetrating steel pins. One LGis exposed. The duct and gland is freed via blunt dissection and care istaken not to injure nearby nerves and blood vessels. The thin connectivetissue capsule enclosing the gland is carefully opened and removed. Tolimit the spread of the superfusate, the LG is covered with a Kimwipemesh. Fluid is drained from the mesh with a rigid aspiration pipette.Drugs and chemicals are added to the superfusate with a wash-in time of˜30 seconds. A microcapillary tube is placed near the lacrimal duct tocollect basal and stimulated tear fluid. After fluid from one gland hasbeen collected the other gland is similarly exposed for collection oftear fluid.

A mouse typically yields about 1 μL of tear fluid, varying according tothe age, gender and extent of disease development. All biomarkers inpooled mouse tear samples, from 5 mice per experimental data point, areanalyzed. At least 5 separate assays points are obtained from pooledspecimens under each disease condition or time point. Assays will varyaccording to the biomarker under study. For CtsS, lactoperoxidase, andlysozyme one can measure enzymatic activity while for Apo-F, Lcn-2 andlactoferrin, one can use either Western blotting or ELISA. For Lcn-2measurement can also use the cyclotide-based assay described in previousparagraphs.

Analysis of ocular surface and LG disease: At the conclusion of the tearcollection, eyeballs and LG are isolated and either flash frozen orparaffin-embedded according to standard methods to obtain tissuesections for immunofluorescence microscopy or histology. Sections fromLG are stained with hematoxylin and eosin to evaluate cytoarchitectureand to assess lymphocytic infiltration. If evidence for lymphocyticinfiltration is seen, extent of lymphocytic infiltration are quantifiedper unit area of LG and the identify of infiltrating cells (T cells, Bcells, macrophages) are further assessed using indirectimmunofluorescence of frozen sections and appropriate commercial primaryand secondary antibodies. One can also isolate whole LG and measurelevels of inflammatory cytokines using RT-PCR as evidence ofinflammatory disease. Corneal and conjunctival sections from control anddisease model mice are processed with Movat's pentachrome stain orPeriodic-Schiff's reagent to detect loss of goblet cells and mucins andcorneal stromal damage, both of which are established as markers forocular surface damage (Dursun et al. (2002) Adv Exp Med. Biol. 506(PtA):647-55). The same sections can be analyzed by hematoxylin and eosinstaining to evaluate possible lymphocytic infiltration, and if seen, thecomposition of the infiltrates can be analyzed by indirectimmunofluorescence as above.

Measurement of biomarkers in tears and LG: Activity (CtsS,Lactoperoxidase, Lysozyme) or protein content (Apo-F, lactoferrin,Lcn-2) can be measured as described for each protein in the precedingbiomarkers section, and signal normalized both to tear volume collectedas well as total tear protein. This latter calculation is particularlyimportant since in some of the primary SjS patients, tears may need tobe collected by gentle washing of the ocular surface because of thecomplete loss of fluid flow. To understand how changes in tearcomposition may be due to disease-specific changes in gene expression ofthe proteins, one can conduct real-time PCR analysis of the relativemRNA content for each protein. This will help in determining the mostspecific biomarkers that are most directly associated with severeinflammatory disease.

Interpretation and Challenges

Applicants predict that the abundance of each of the disease-inducedbiomarkers (CtsS, Apo-F and Lcn-2) in tears from control animals will beminimal, and that their content in the tears will increase in proportionto the severity of autoimmune inflammatory disease in the mouse models.It has never been explored whether the expression of these biomarkersincreases as disease progresses in the NOD mouse, beyond the 4-12 weektime-points measured so far at disease onset. It has also never beenexplored whether these biomarkers are secreted into tears in any othermouse model of disease. These potential novel biomarkers are newlydiscovered and have also never been comparatively measured in healthyand primary SjS patient tears. The parallel measurement of geneexpression using real-time PCR will aid Applicants in identifyingmarkers that are uniquely upregulated and secreted to tears ininflammatory autoimmune disease. Such an increase over a low backgroundor threshold would be ideal for translational development as a biomarkerof disease.

Experiment 3

Purpose: Previous work has revealed age- and inflammatorydisease-correlated alterations in apolipoproteins F (Apo-F) and E(Apo-E) in mouse lacrimal gland (LG) along with abnormal lipiddeposition. The current experiment characterizes the expression anddistribution of apolipoproteins in human LG in comparison to healthy(BALB/c) and Sjögren's syndrome disease model (NOD) mouse LG.

Methods: Microarray was conducted with RNAs prepared from two human maleLG groups, young (54 years average) and old (77 years average) (n=3 pergroup), and data compared using Partek GS software (Parteck Inc. St.Louis, Mo.). Cryosections of LGs from humans of both genders and mousemodels were evaluated microscopically using immunofluorescence and OilRed O.

Results: Microarray analysis detected expression of the following genesin human LG: APO-C1, APO-D, APO-E and APO-F. In older males, APO-C1,APO-D and APO-E genes were moderately upregulated to 2.6, 1.5 and 1.3folds while APO-F was downregulated to 60% relative to the younger group(p<0.02). Apo-E protein was universally detected at the basolateralmembrane of acinar cells from different specimens. However, Apo-Fprotein distribution varied markedly in acinar cells. Apo-F was observedin some human LGs at the cytoplasmic face of the plasma membrane butbeneath basolateral ApoE, rather than the co-localization of the twoproteins seen in BALB/c mouse acinar cells. ApoF in acinar cells fromother human LG was detected in a dispersed distribution withincytoplasmic and secretory vesicle-like organelles comparable to thepattern in NOD mouse LG. Without being bound by theory, this findingindicates that similar to mouse, human individuals in which Apo-F isdispersedly distributed within cyoplasmic and secretory vesicle-likeorganelles are likely suffering from Sjögren's syndrome or at risk ofdeveloping Sjögren's syndrome, whereas human individuals in which Apo-Fis located at the cytoplasmic face of the plasma membrane in the acinarcells are likely not suffering from Sjögren's syndrome or at risk ofdeveloping Sjögren's syndrome. The dispersed distribution of ApoF in NODmouse LG and certain human LG is believed to result from missorting ofApoF in the acinar cells. Finally, Oil Red O staining showed manyadipocytes scattered within human LG but not within mouse LG.

Conclusion: The detection of adipocytes and several apolipoprotein mRNAsindicates that the human LG is active in lipid metabolism. Apo-F issubject to age-related regulation and its cellular location appearshighly variable in human LG, suggesting the possibility of alterationsunder pathological conditions. This finding further indicates that, thedisparate distribution patterns of missorted proteins, such as Apo-F aswell as CATS as observed in the NOD mouse model compared to healthymice, are promising markers for the diagnosis of Sjögren's syndrome, notonly in mouse, but also in human and other mammals. Accordingly, thepresence of Apo-F or CATS at the cytoplasmic face of the plasma membranein acinar cells in a LG from a mammal indicates that the mammal is notlikely suffering from Sjögren's syndrome or is at risk of developingSjögren's syndrome. A dispersed distribution of Apo-F or CATS withincytoplasmic or secretory vesicle-like organelles in acinar cells in a LGfrom a mammal indicates that the mammal is suffering from Sjögren'ssyndrome or is at risk of developing Sjögren's syndrome.

Experiment 4

This experiment was designed to determine the range of activity levelsof cathepsin S in normal and diseased human tears using a simple,accessible assay based on the Schirmer's test that is available to thenon-specialist. Meanwhile, the experiment determines if increasedcathepsin S activity per unit protein in tears is correlated withdiagnosed Sjögren's syndrome, so that it may function as a diagnosticbiomarker.

A Schirmer's test, which measures tear flow, is a routine part of theintake process for all new patients and for the continuing evaluation ofpatients with ocular surface disorders. Classification for a Schirmer'stest is as follows. A Schirmer's value 0-5 mm indicates severe dry eye,5.1-10 mm indicates moderate dry eye, 10.1-15 mm indicates mild dry eye,and >15 mm indicates normal tear flow.

All patients receiving Schirmer's tests by the participating physicianswere given an informed consent form and a small recognition forparticipation.

Fifty-three patients (106 eyes) were tested (all-corners trial) frompatients accrued at the USC/Doheny Ophthalmology Clinic. Schirmer'sstrips, which measure unstimulated wetting of the anesthetized eye over5 min, were collected and stored for up to 4 hours at 4° C. Humancathepsin S catalytic activity eluted from Schirmer's strips does notchange within this window. Assays are conducted blinded (samples werelabeled with regards to left or right eye). The proteins are eluted fromSchirmer's strips and assayed for cathepsin S activity and for proteincontent.

Patient gender and disease status are then provided and matched to theactivity assay values for analysis of general trends.

As shown in FIG. 10, female patients tend to have higher CATS activitiesthan male. FIG. 11 shows that a number of patients showed drasticallyincreased CATS activities. Surprisingly, two of the three patients withthe highest CATS activities were diagnosed with Sjögren's syndrome andlupus.

FIG. 12 shows that high CATS activity correlated with SjS and lupusconditions (CATS activity for the diagnosed patients<11831). It is worthnoting that other patients exhibiting high CATS/protein activity mayhave undiagnosed SjS/lupus.

Further, FIGS. 13 and 14 show bar charts indicating that the CATScaptivities in SjS and lupus patients were significantly higher thanother patients or patients with other diseases.

It was further observed that patients with lower Schirmer's valuesgenerally had higher CATS activities (FIGS. 15-24) and older malepatients generally had higher CATS activities (FIGS. 27-28). The agedifference among female patients, however, was not observed (FIGS.25-26).

Therefore, the current experiment shows that (1) female CtsS/protein intears higher than in males; (2) in both males and females, CtsS/proteinin tears is highest in patients with low Schirmer's scores; (3) the twopatients with diagnosed SjS or lupus had the highest CtsS/proteinactivities.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

1. A method for one or more of: a). determining whether a mammal is or is not likely or not likely to develop autoimmune disease; or b). aiding in diagnosing an autoimmune disease in a mammal; or c). diagnosing relative severity of an autoimmune disease in a mammal, comprising measuring an expression level or activity level of at least one first polypeptide selected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10, Il10ra, Il15, Tnfa, Apo-F, or Lcn-2, and/or at least one second polypeptide selected from the group lactoperoxidase, lactoferrin or lysozyme in a sample of tear isolated from the mammal, wherein an increased expression level or increased activity level of the first polypeptide or a decreased expression level or decreased activity level of the second polypeptide, as compared to a suitable control, is an indication of one or more of: d). that the mammal is likely to develop autoimmune disease; or e). a likely positive diagnosis of autoimmune disease for the mammal; or f), that the mammal has a relatively more severe autoimmune disease; and a lack of increased expression level or increased activity level of the first polypeptide and a lack of decreased expression level or decreased activity level of the second polypeptide, as compared to a suitable control, indicates that the mammal is an indication of one or more of: q). the mammal is not likely to develop autoimmune disease; or h). a likely negative diagnosis of autoimmune disease for the mammal. 2.-3. (canceled)
 4. The method of claim 1, wherein the first polypeptide is selected from the group Ctss, Apo-F or Lcn-2.
 5. The method of claim 1, wherein the expression level of the polypeptide is measured by one or more method of the group of fluorometric analysis, Western blot, gel electrophoresis, ELISA, mass spectrometry, or protein array.
 6. The method of claim 1, wherein the activity level of a polypeptide is measured by protease activity assay.
 7. The method of claim 1, further comprising examining the mammal with a Schirmer test, a slit-lamp examination, a radiological test, to assist diagnosis.
 8. A method of treating a mammal identified as suffering from or at risk of developing an autoimmune disease and identified as being in need of such treatment by the method of claim 1, comprising administering an effective amount of at least one of the group cyclosporin, cevimeline, pilocarpine, a nonsteroidal anti-inflammatory drug, a corticosteroid, an immunosuppressive drug or a disease-modifying antirheumatic drug, to the mammal, thereby treating the mammal.
 9. The method of claim 1 or 8, wherein the autoimmune disease is one or more selected from the group consisting of Coeliac disease, diabetes mellitus type 1 (IDDM), lupus erythematosus, systemic lupus erythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis (RA), ankylosing spondylitis, Crohns disease, dermatomyositis, Goodpasture's syndrome, Guillain-Barré syndrome (GBS), mixed Connective tissue disease, multiple sclerosis, myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, temporal arteritis, ulcerative colitis, vasculitis and Wegener's granulomatosis.
 10. The method of claim 9, wherein the autoimmune disease is lupus erythematosus or Sjögren's syndrome.
 11. The method of claim 8, wherein the Sjögren's syndrome is an inflammatory autoimmune lacrimal gland disease.
 12. The method of claim 1 or 8, wherein the mammal is a human patient.
 13. A method for treating a mammal identified by the method of claim 1 as suffering from or at risk of developing an autoimmune disease, comprising administering to the mammal an effective amount of an agent inhibiting the expression or activity of a polypeptide selected from the group Ctss, Ctsh, Ctsr, Ctsw or Ctsz.
 14. The method of claim 13, wherein the polypeptide is Ctss.
 15. The method of claim 13 or 14, wherein the agent is a Ctss antibody.
 16. The method of claim 13 or 14, wherein the agent is a small molecule Ctss inhibitor.
 17. A kit to diagnose autoimmune disease, comprising a probe or an antibody that is specific for a polypeptide selected from the group Ctss, Ctsh, Ctsr, Ctsw, Ctsz, Ifng, Il6ra, Il10, Il10ra, Il15, Tnfa, Apo-F, Lcn-2, lactoperoxidase, lactoferrin or lysozyme, and instructions to use.
 18. The kit of claim 17, further comprising a Schirmer's test strip to collect human (patient) tears.
 19. A method for determining whether a mammal is likely or is not likely to develop autoimmune disease or aiding in the diagnosis of an autoimmune disease, comprising determining the presence of a protein selected from CATS or APO-F in an acinar cell isolated from the lacrimal gland or salivary gland of the mammal, wherein the mammal is likely to develop the autoimmune disease if more than about 20% of the protein in the cell is present not in proximity to the basolateral membrane of the cell or the mammal is not likely to develop the autoimmune disease if more than about 80% of the protein in the cell is present in proximity to the basolateral membrane of the cell.
 20. The method of claim 19, wherein the mammal is likely to develop the autoimmune disease if more than about 50% of the protein in the cell is present not in proximity to the basolateral membrane of the cell.
 21. A method for aiding in diagnosing autoimmune disease in a mammal, comprising determining the presence of a protein selected from CATS or APO-F in an acinar cell isolated from the lacrimal gland or salivary gland of the mammal, wherein presence of more than about 20% of the protein in the cell not in proximity to the basolateral membrane of the cell indicates a likely positive diagnosis of the autoimmune disease for the mammal, thereby aiding in the diagnosis or presence of more than about 80% of the protein in the cell in proximity to the basolateral membrane of the cell indicates a likely negative diagnosis of the autoimmune disease for the mammal, thereby aiding in the diagnosis.
 22. The method of claim 19, wherein presence of more than about 50% of the protein in the cell not in proximity to the basolateral membrane of the cell indicates a likely positive diagnosis of the autoimmune disease for the mammal, thereby aiding in the diagnosis.
 23. The method of claim 19, wherein the protein is APO-F.
 24. The method of claim 19, wherein the autoimmune disease is one or more selected from the group consisting of Coeliac disease, diabetes mellitus type 1 (IDDM), lupus erythematosus, systemic lupus erythematosus (SLE), Sjögren's syndrome, Churg-Strauss Syndrome, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis (RA), ankylosing spondylitis, Crohns disease, dermatomyositis, Goodpasture's syndrome, Guillain-Barré syndrome (GBS), mixed Connective tissue disease, multiple sclerosis, myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anaemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, temporal arteritis, ulcerative colitis, vasculitis and Wegener's granulomatosis.
 25. The method of claim 24, wherein the autoimmune disease is lupus erythematosus or Sjögren's syndrome.
 26. The method of claim 19, wherein the mammal is a human.
 27. The method of any one of claims 1, 8 or 19 wherein measurement of the expression level or activity level of the one first and/or second polypeptide comprises collecting a tear sample from the patient on a Schirmer's test strip containing a quantitative and detectably labeled substrate specific for the first and/or second polypeptide.
 28. The method of claim 27, wherein the quantitative detectably labeled substrate is a fluorometrically labeled antibody. 